Capture probes for use in detecting the presence of Trichomonas vaginalis in a sample

ABSTRACT

Oligomers useful for determining the presence of  Trichomonas vaginalis  in a test sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/011,811, filed Jan. 21, 2011, now U.S. Pat. No. 8,790,879, whichclaims the benefit of U.S. Provisional Application No. 61/297,367, filedJan. 22, 2010, the contents of each of which applications i-s are herebyincorporated by reference herein in its entirety.

FIELD

The present disclosure relates to detection probes, capture probes,amplification oligonucleotides, nucleic acid compositions, probe mixes,methods, and kits useful for determining the presence of Trichomonasvaginalis in a test sample.

BACKGROUND

Trichomonas vaginalis is protozoan parasite that causes trichomoniasis,one of the most common and treatable of the sexually transmitteddiseases. Trichomonas vaginalis is a relatively delicate pear-shapedtrophozoite that is typically 7 to 23 μm long by 5 to 12 μm wide. Theorganism has four anterior flagella and a fifth forming the outer edgeof a short undulating membrane. The anterior flagella propels theorganism through liquid in a jerky, rapid fashion, sometimes causing theorganism to rotate as it moves. Trichomonas vaginalis divides by binaryfission in the urogenital tract of those infected. The organism istranslucent and colorless, or slightly grey in appearance under themicroscope. A slender rod, the axostyle, extends the length of the bodyand protrudes posteriorly. The nucleus is near-anterior and appearswell-defined, containing many chromatin granules. The appearance of T.vaginalis is very similar to that of other trichomonads, such asTrichomonas tenax, although only T. vaginalis is found in genitourinarytract infections.

Worldwide, T. vaginalis infects approximately 180 million people peryear, usually by direct person-to-person contact, making it the mostcommon sexually transmitted disease (STD) agent. In the United States,it is believed that T. vaginalis infects an estimated 7 million peopleannually. Despite its prevalence and geographic distribution, T.vaginalis has not been the focus of intensive study. Indeed, it is noteven listed as a “reportable disease” by the U.S. Centers for DiseaseControl, and there are no active control or prevention programs. Recentreports, however, suggest growing public health interest in thispathogen. Infections in women are known to cause vaginitis, urethritis,and cervicitis. Severe infections are accompanied by a foamy,yellowish-green discharge with a foul odor, and small hemorrhagiclesions may also be present in the genitourinary tract. Complicationsinclude premature labor, low-birth weight offspring, premature ruptureof membranes, and post-abortion and post-hysterectomy infection. Anassociation with pelvic inflammatory disease, tubal infertility, andcervical cancer have been reported. Trichomonas vaginalis has also beenimplicated as a co-factor in the transmission of HIV and other STDagents. The organism can also be passed to neonates during passagethrough the birth canal.

In men, symptoms of trichomoniasis include urethral discharge, urethralstricture, epididymitis, the urge to urinate, and a burning sensationwith urination. In both men and women, infections with T. vaginalis areusually asymptomatic and may be self-limiting. It is estimated that, inwomen, 10-50% of T. vaginalis infections are asymptomatic, with theproportion in men probably being even higher. That said, with many womenthe infection becomes symptomatic and chronic, with periods of relief inresponse to therapy. Recurrence may be caused by re-infection from anasymptomatic sexual partner, or by failure of the standard course oftherapy (a regimen of the antibiotic metronidazole). And while T.vaginalis infections almost always occur in the genitourinary tract, onrare occasions they occur at ecotopic sites, and the parasite may berecovered from other areas of a patient's body.

As a result of suboptimal comparative laboratory methods and a focus onother STD sources, studies of T. vaginalis have often substantiallyunderestimated the prevalence of infection. Despite this, levels ofinfection typically have been high, with reported overall prevalencerates ranging from 3-58%, with an unweighted average across studies of21% (Cu-Uvin et al. Clin. Infect. Dis. (2002) 34(10):1406-11). Instudies that presented information on race/ethnicity, T. vaginalisinfection rates have been reported to be highest among African-Americans(Sorvillo et al. Emerg. Infect. Dis. (2001) 7(6):927-32). The followingchart illustrates the trend reported by Sorvillo et al., with regard tothe prevalence of infection in terms of the percentage of patientsinfected with trichomoniasis, chlamydia, and/or gonorrhea at certainhealth clinics in Baltimore, Md. (B) and in New York, N.Y. (NY).

Patient Trichomoniasis Chlamydia Gonorrhea Year Number City (%) (%) (%)1996 213 NY 51 9 5 1994 372 NY 27 7 2 1994 1404 NY 20 15 No Data 1992279 B 26 21 14  1990-94 677 NY 22 6 1

Following exposure, the incubation period ranges from about 5 to 10days, although periods as short as 1 day to as many as 28 days have beenreported. If diagnosed, T. vaginalis infections can be readily treatedby orally administered antibiotics.

Given its relative prevalence and association with other STDs, there isincreasing interest in effectively diagnosing trichomoniasis.Conventional diagnostic methods for detecting T. vaginalis, however, arebased on direct examination, “wet mount” microscopy, or cell culture,each of which has its own shortcomings. With regard to direct patientexamination, other infections mimic the appearance and odor of thevaginal discharge. Accordingly, laboratory techniques such asmicroscopy, antibody detection, and cell culture are often used. Whileit is possible to detect T. vaginalis using a “wet mount” prepared bymixing vaginal secretions with saline on a slide and examining the slideunder a microscope for the presence of organisms having thecharacteristic size, shape, and motility of T. vaginalis, thesensitivity of such methods depends highly on the skill and experienceof the microscopist, as well as the time spent transporting specimen toa laboratory. Wet mount diagnosis has been found to be only 35-80% assensitive as other methods, such as cell culture, in detecting thepresence of T. vaginalis. Other direct methods, such as fluorescentantibody detection and enzyme-linked immunoassays, have also beendeveloped, as has a non-amplified, DNA probe-based method (Affirm,Becton Dickinson), although their sensitivities, as compared to cellculture, range from 70-90%. For these reasons, cell culture isconsidered the current “gold standard” for clinical detection of T.vaginalis. Due to its relatively delicate nature, however, culturing theorganism is technically challenging, and typically requires up to 7 daysfor maximum sensitivity. Even then, the sensitivity of cell culturemethods is estimated to be only about 85-95% due to problems associatedwith time lapses between sample recovery and culture inoculation,maintaining proper incubation conditions, visualizing low numbers of theorganism and/or the motility of the protozoa.

Given the human health implications of trichomoniasis and relativeinability of existing clinical laboratory methods to selectively andsensitively detect T. vaginalis from a test sample, a need clearlyexists for a sensitive and specific assay which can be used to determinethe presence of T. vaginalis in a particular sample of biologicalmaterial.

SUMMARY

The present disclosure provides a solution to the clinical need for asensitive assay specific for T. vaginalis by featuring oligonucleotidesthat are useful for determining whether T. vaginalis is present in atest sample, such as a genitourinary specimen.

Detection probes are provided that preferentially hybridize to a targetregion present in nucleic acid derived from T. vaginalis to form adetectable probe:target hybrid indicating the presence of T. vaginalis.In one embodiment, the disclosure provides detection probes fordetermining whether T. vaginalis is present in a test sample derivedfrom a biological material obtained from, for example, the genitourinarytract of a patient. The detection probes contain a target-complementarybase sequence that is perfectly complementary to a target sequencecontained within a target domain derived from T. vaginalis, where thetarget domain is selected from the group consisting of (reading 5′ to3′):

SEQ ID NO: 37 ttgccgaagtccttcggttaaagttctaattgggactccctgcg,SEQ ID NO: 38 uugccgaaguccuucgguuaaaguucuaauugggacucccugcg,SEQ ID NO: 39 cgcagggagtcccaattagaactttaaccgaaggacttcggcaa,SEQ ID NO: 40 cgcagggagucccaauuagaacuuuaaccgaaggacuucggcaa,and RNA/DNA combination equivalents of the foregoing. Thetarget-complementary base sequence of the detection probes includes thebase sequence of (reading 5′ to 3′):

SEQ ID NO: 41 ttcggttaaagttctaa, SEQ ID NO: 42 uucgguuaaaguucuaa,SEQ ID NO: 43 ttagaactttaaccgaa, SEQ ID NO: 44 uuagaacuuuaaccgaa,and RNA/DNA combination equivalents of the foregoing.

In one embodiment, the detection probes contain a target-complementarybase sequence having a base sequence selected from the group consistingof (reading 5′ to 3′):

SEQ ID NO: 1 ttgccgaagtccttcggttaaagttctaattg, SEQ ID NO: 2uugccgaaguccuucgguuaaaguucuaauug, SEQ ID NO: 3caattagaactttaaccgaaggacttcggcaa, SEQ ID NO: 4caauuagaacuuuaaccgaaggacuucggcaa, SEQ ID NO: 5tgccgaagtccttcggttaaagttctaattgg, SEQ ID NO: 6ugccgaaguccuucgguuaaaguucuaauugg, SEQ ID NO: 7ccaattagaactttaaccgaaggacttcggca, SEQ ID NO: 8ccaauuagaacuuuaaccgaaggacuucggca, SEQ ID NO: 9gccgaagtccttcggttaaagttctaattggg, SEQ ID NO: 10gccgaaguccuucgguuaaaguucuaauuggg, SEQ ID NO: 11cccaattagaactttaaccgaaggacttcggc, SEQ ID NO: 12cccaauuagaacuuuaaccgaaggacuucggc, SEQ ID NO: 13ccgaagtccttcggttaaagttctaattggg, SEQ ID NO: 14ccgaaguccuucgguuaaaguucuaauuggg, SEQ ID NO: 15cccaattagaactttaaccgaaggacttcgg, SEQ ID NO: 16cccaauuagaacuuuaaccgaaggacuucgg, SEQ ID NO: 17cgaagtccttcggttaaagttctaattgggac, SEQ ID NO: 18cgaaguccuucgguuaaaguucuaauugggac, SEQ ID NO: 19gtcccaattagaactttaaccgaaggacttcg, SEQ ID NO: 20gucccaauuagaacuuuaaccgaaggacuucg, SEQ ID NO: 21cgaagtcittcggttaaagttctaattgggac, SEQ ID NO: 22cgaaguciuucgguuaaaguucuaauugggac, SEQ ID NO: 23gtcccaattagaactttaaccgaaigacttcg, SEQ ID NO: 24gucccaauuagaacuuuaaccgaaigacuucg, SEQ ID NO: 25gaagtccttcggttaaagttctaa, SEQ ID NO: 26 gaaguccuucgguuaaaguucuaa,SEQ ID NO: 27 ttagaactttaaccgaaggacttc, SEQ ID NO: 28uuagaacuuuaaccgaaggacuuc, SEQ ID NO: 29 gtccttcggttaaagttctaattgg,SEQ ID NO: 30 guccuucgguuaaaguucuaauugg, SEQ ID NO: 31ccaattagaactttaaccgaaggac, SEQ ID NO: 32 ccaauuagaacuuuaaccgaaggac,SEQ ID NO: 33 ttcggttaaagttctaattgggactccctgcg, SEQ ID NO: 34uucgguuaaaguucuaauugggacucccugcg, SEQ ID NO: 35cgcagggagtcccaattagaactttaaccgaa, SEQ ID NO: 36cgcagggagucccaauuagaacuuuaaccgaa,and RNA/DNA combination equivalents of the foregoing.

In the present disclosure, the detection probes may have atarget-complementary base sequence of any length suitable to achieve thedesired selectivity and specificity for T. vaginalis-derived nucleicacid. Detection probes of the present disclosure comprise anoligonucleotide up to 100 bases in length in one embodiment, or are from25 to 50 bases in length in another embodiment, or are from 25 to 35bases in length in yet another embodiment. In one embodiment, thetarget-complementary base sequence of the detection probes is perfectlycomplementary to the target sequence.

In one embodiment, the base sequence of the detection probe consists ofa target-complementary base sequence contained within and comprising atleast 25 contiguous bases of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,SEQ ID NO:36, or an RNA/DNA combination equivalent of any of theforegoing, and, optionally, one or more base sequences that arenon-complementary to a nucleic acid derived from T. vaginalis. Anadditional base sequence may be comprised of any desired base sequence,so long as it does not stably bind to nucleic acid derived from the T.vaginalis under stringent hybridization conditions or prevent stablehybridization of the probe to the target nucleic acid. By way ofexample, additional bases may be included if the base sequence of thetarget-complementary base sequence is incorporated into a “molecularbeacon” probe. Molecular beacons are disclosed by Tyagi et al.,“Detectably Labeled Dual Conformation Oligonucleotide Probes, Assays andKits,” U.S. Pat. No. 5,925,517, (the contents of which are herebyincluded by reference herein), and include a target-complementary basesequence which is bounded by two base sequences having regions which areat least partially complementary to each other. A more detaileddescription of molecular beacons is provided infra in the sectionentitled “Hybridization Assay Probes to T. vaginalis Ribosomal NucleicAcid.” An additional base sequence may be joined directly to thetarget-complementary base sequence or, for example, by means of anon-nucleotide linker.

Detection probes according to the disclosure form a probe:target hybridstable for detection with nucleic acid derived from T. vaginalis understringent conditions and does not form a hybrid stable for detectionwith nucleic acid derived from Trichomonas tenax (ATCC® No. 30207) understringent conditions.

The target-complementary base sequence may consist of deoxyribonucleicacid (DNA), ribonucleic acid (RNA), a combination of DNA and RNA, or itmay be a nucleic acid analog (e.g., a peptide nucleic acid) or containone or more modified nucleosides (e.g., a ribonucleoside having a2′-O-methyl substitution to the ribofuranosyl moiety). Thetarget-complementary base sequence may additionally include moleculesthat do not hydrogen bond to adenine, cytosine, guanine, thymine oruracil, provided such molecules do not interfere with the ability of thedetection probe to selectively and specifically bind to nucleic acidderived from T. vaginalis in the test sample. Such molecules couldinclude, by way of example, abasic nucleotides or universal baseanalogues, such as 5-nitroindole, provided such molecules do notsignificantly affect duplex stability. See, e.g., Guo et al.,“Artificial Mismatch Hybridization,” U.S. Pat. No. 5,780,233, thecontents of which are incorporated by reference herein.

Detection probes of the present disclosure may include a detectablelabel. The label may be any suitable labeling substance, including butnot limited to a radioisotope, an enzyme, an enzyme cofactor, an enzymesubstrate, a dye, a hapten, a chemiluminescent molecule, a fluorescentmolecule, a phosphorescent molecule, an electrochemiluminescentmolecule, a chromophore, a base sequence region that is unable to stablyhybridize to the target nucleic acid under the stated conditions, andmixtures of these. In one particular embodiment, the label is anacridinium ester (AE), for example, 4-(2-succinimidyloxycarbonylethyl)-phenyl-10-methylacridinium-9-carboxylate fluorosulfonate(hereinafter referred to as “standard AE”) or9[[4-[3-[(2,5-dioxo-1-pyrrolidinyl)oxy]-3-oxopropyl]phenoxy]carbonyl]-2,s10-dimethyl-acridiniumtrifluoromethane sulfonate (hereinafter referred to as “glower AE”).Detection probes of the present disclosure may also include groups ofinteracting labels. Such groups of interacting labels include, by way ofexample, the following groups: enzyme/substrate, enzyme/cofactor,luminescent/quencher, luminescent/adduct, dye dimers and Forresterenergy transfer pairs.

In one embodiment of the present disclosure, a capture probe is providedfor extracting a T. vaginalis-derived target nucleic acid present in atest sample. The base sequence of the capture probe consists of atarget-complementary base sequence that is perfectly complementary to atarget sequence contained within a target domain selected from the groupconsisting of (reading 5′ to 3′):

SEQ ID NO: 77 gtgcgtgggttgacctgtctagcgttgatt, SEQ ID NO: 78gugcguggguugaccugucuagcguugauu, SEQ ID NO: 79aatcaacgctagacaggtcaacccacgcac, SEQ ID NO: 80aaucaacgcuagacaggucaacccacgcac,and RNA/DNA combination equivalents of the foregoing, and, optionally,at least one base sequence that is non-complementary to the T.vaginalis-derived target nucleic acid. The target-complementary basesequence of the capture probe includes the base sequence of (reading 5′to 3′):

SEQ ID NO: 81 gacctgtcta, SEQ ID NO: 82 gaccugucua, SEQ ID NO: 83tagacaggtc, SEQ ID NO: 84 uagacagguc,or an RNA/DNA combination equivalent of any of the foregoing.

In another embodiment, the target-complementary base sequence of thecapture probe includes a base sequence selected from the groupconsisting of (reading 5′ to 3′):

SEQ ID NO: 55 gcctgctgctacccgtggatat SEQ ID NO: 56gccugcugcuacccguggauau SEQ ID NO: 57 atatccacgggtagcagcaggcSEQ ID NO: 58 auauccacggguagcagcaggc SEQ ID NO: 85ctagacaggtcaacccacgcac, SEQ ID NO: 86 cuagacaggucaacccacgcac,SEQ ID NO: 87 gtgcgtgggttgacctgtctag, SEQ ID NO: 88gugcguggguugaccugucuag, SEQ ID NO: 90 aatcaacgctagacaggtcaaccc,SEQ ID NO: 91 aaucaacgcuagacaggucaaccc, SEQ ID NO: 92gggttgacctgtctagcgttgatt, SEQ ID NO: 93 ggguugaccugucuagcguugauu,SEQ ID NO: 95 tcaacgctagacaggtcaa, SEQ ID NO: 96 ucaacgcuagacaggucaa,SEQ ID NO: 97 ttgacctgtctagcgttga, SEQ ID NO: 98 uugaccugucuagcguuga,SEQ ID NO: 100 aatcaacgctagacaggtc, SEQ ID NO: 101 aaucaacgcuagacagguc,SEQ ID NO: 102 gacctgtctagcgttgatt, SEQ ID NO: 103 gaccugucuagcguugauu,and RNA/DNA combination equivalents of the foregoing.

Capture probes according to the present disclosure may be immobilized ona solid support by means of ligand-ligate binding pairs, such asavidin-biotin linkages, but may also include an immobilized probebinding region. The immobilized probe binding region of the captureprobes is comprised of any base sequence capable of stably hybridizingunder assay conditions to an oligonucleotide that is bound to a solidsupport present in a test sample. In one embodiment, the immobilizedprobe binding region is a poly dA, homopolymer tail positioned at the 3′end of the capture probe. In this embodiment, oligonucleotides bound tothe solid support would include 5′ poly dT tails of sufficient length tostably bind to the poly dA tails of the capture probes under assayconditions. In another embodiment, the immobilized probe binding regionincludes a poly dA tail which is about 30 adenines in length, and thecapture probe includes a spacer region which is about 3 thymines inlength for joining together the target-complementary base sequence andthe immobilized probe binding region (SEQ ID NO: 50tttaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa).

The present disclosure also features amplification oligonucleotidesuseful for determining the presence of T. vaginalis in an amplificationassay. In one embodiment, the disclosure provides at least oneamplification oligonucleotide for amplifying nucleic acid derived fromT. vaginalis present in a test sample, where the base sequence of theamplification oligonucleotide consists of a 3′ target-complementary basesequence up to 40 bases in length and containing the base sequence of:

SEQ ID NO: 45 gctaacgagcgagattatcgcc, SEQ ID NO: 46gcuaacgagcgagauuaucgcc, SEQ ID NO: 47 ggcgataatctcgctcgttagc,SEQ ID NO: 48 ggcgauaaucucgcucguuagc, SEQ ID NO: 49ggcatcacggacctgttattgc, SEQ ID NO: 50 ggcaucacggaccuguuauugc,SEQ ID NO: 51 gcaataacaggtccgtgatgcc, SEQ ID NO: 52gcaauaacagguccgugaugcc,or an RNA/DNA combination equivalent of any of the foregoing and,optionally, includes a sequence located 5′ to the 3′target-complementary base sequence that is non-complementary to a T.vaginalis derived nucleic acid. The optional sequence may be, forexample, a sequence recognized by an RNA polymerase or which enhancesinitiation or elongation by RNA polymerase, such as the T7 promotersequence of SEQ ID NO:54: aatttaatacgactcactatagggaga.

In another embodiment, the amplification oligonucleotides are employedin sets of at least two amplification oligonucleotides. In one set, afirst amplification oligonucleotide is included, the base sequence ofwhich consists of a 3′ target-complementary base sequence up to 40 basesin length and containing the base sequence of SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, or an RNA/DNA combination equivalentof any of the foregoing and, optionally, a 5′ sequence that isnon-complementary to a T. vaginalis derived nucleic acid. The basesequence of a second amplification oligonucleotide of the set consistsof a 3′ target-complementary base sequence up to 40 bases in length andcontaining the base sequence of SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, or an RNA/DNA combination equivalent of any of theforegoing and, optionally, a 5′ sequence that is non-complementary to aT. vaginalis derived nucleic acid. The optional sequence may be, forexample, a sequence recognized by an RNA polymerase or which enhancesinitiation or elongation by RNA polymerase, such as the T7 promotersequence of SEQ ID NO:54.

The present disclosure, also a relates to probe mixes for determiningthe presence of T. vaginalis in a test sample. In one embodiment, theprobe mix includes at least one of the above-described detection probesand at least one of the above-described capture probes. In anotherembodiment, the probe mix includes at least one of the above-describeddetection probes and at least one of the above-described amplificationoligonucleotides. In another embodiment, the probe mix includes at leastone of the above-described detection probes, at least one of theabove-described capture probes, and at least one of the above-describedamplification oligonucleotides. In another embodiment, the probe mixincludes at least one of the above-described detection probes, at leastone of the above-described capture probes, and at least two of theabove-described amplification oligonucleotides. In another embodiment,the probe mix includes at least one of the above-described detectionprobes and at least two of the above-described capture probes. Inanother embodiment, the probe mix includes at least one of theabove-described detection probes and at least two of the above-describedamplification oligonucleotides. In another embodiment, the probe mixincludes at least one of the above-described detection probes, at leasttwo of the above-described capture probes, and at least one of theabove-described amplification oligonucleotides. In another embodiment,the probe mix includes at least one of the above-described detectionprobes, at least two of the above-described capture probes, and at leasttwo of the above described amplification oligonucleotides.

The present disclosure further features methods for determining whetherT. vaginalis is present in a test sample. In one embodiment, the methodcomprises the steps of contacting the test sample with at least one ofthe above-described detection probes for detecting T. vaginalis understringent conditions, and determining whether the probe:target hybridhas formed as an indication of the presence or absence of T. vaginalisin the test sample. This method may further include the step ofquantifying the amount of hybrid present in the test sample as a meansfor estimating the amount of T. vaginalis present in the test sample. Inanother embodiment, the method comprises contacting the test sample withat least one of the above-described detection probes and at least one ofthe above-described capture probes under stringent conditions. Inanother embodiment, the method comprises contacting the test sample withat least one of the above-described detection probes under stringentconditions, and at least one of the above described amplificationoligonucleotides under amplification conditions. In another embodiment,the method comprises contacting the test sample with at least one of theabove-described detection probes and at least one of the above describedcapture probes under stringent conditions, and at least one of theabove-described amplification oligonucleotides under amplificationconditions. In another embodiment, the method comprises contacting thetest sample with at least one of the above-described detection probesand at least one of the above described capture probes under stringentconditions, and at least two of the above-described amplificationoligonucleotides under amplification conditions. In another embodiment,the method comprises contacting the test sample with at least one of theabove-described detection probes and at least two of the above-describedcapture probes under stringent conditions. In another embodiment, themethod comprises contacting the test sample with at least one of theabove-described detection probes under stringent conditions, and leasttwo of the above-described amplification oligonucleotides underamplification conditions. In another embodiment, the method comprisescontacting the test sample with at least one of the above-describeddetection probes and at least two of the above-described capture probesunder stringent conditions, and at least one of the above-describedamplification oligonucleotides under amplification conditions. Inanother embodiment, the method comprises contacting the test sample withat least one of the above-described detection probes and at least two ofthe above-described capture probes under stringent conditions, and atleast two of the above-described amplification oligonucleotides underamplification conditions.

The disclosure also contemplates kits for determining whether T.vaginalis is present in a test sample. These kits comprise at least oneof the above-described detection probes specific for T. vaginalisderived nucleic acid and optionally include written instructions fordetermining the presence or amount of T. vaginalis in a test sample. Inanother embodiment, the kits also include at least one of theabove-described amplification oligonucleotides appropriate foramplifying the target sequence or its complement. In yet anotherembodiment, the kits also include at least one of the above-describedcapture probes for separating the target nucleic acid from othercomponents of the test sample prior to amplifying or directly detectingthe target sequence or its complement.

Those skilled in the art will appreciate that the detection probes ofthe present disclosure may be used as amplification oligonucleotides orcapture probes, the amplification oligonucleotides may be used adetection probes or capture probes, and the capture probes may be usedas amplification oligonucleotides or detection probes depending upon thedegree of specificity required. Other features and advantages of thedisclosure will be apparent from the following description of theembodiments thereof and from the claims.

DESCRIPTION

The present disclosure describes oligonucleotides targeted to nucleicacids derived from T. vaginalis which are particularly useful fordetermining the presence or absence of T. vaginalis in a test sample.The oligonucleotides can aid in detecting T. vaginalis in differentways, such as by functioning as detection probes, capture probes, and/oramplification oligonucleotides. Detection probes of the presentdisclosure can preferentially hybridize to a target nucleic acidsequence present in a target nucleic acid derived from T. vaginalisunder stringent hybridization conditions to form detectable duplexeswhich indicate the presence of T. vaginalis in a test sample. Probes ofthe present disclosure are believed to be capable of distinguishingbetween T. vaginalis and its known closest phylogenetic neighbor.Capture probes of the present disclosure can hybridize to a targetnucleic acid sequence present in nucleic acid derived from T. vaginalisunder assay conditions and can be used to separate target nucleic acidfrom other components of a clinical specimen. Amplificationoligonucleotides of the present disclosure can hybridize to a targetnucleic acid sequence present in nucleic acid derived from T. vaginalisunder amplification conditions and can be used, for example, as primersin amplification reactions to generate multiple copies of T.vaginalis-derived nucleic acid. The probes and amplificationoligonucleotides can be used in assays for the detection and/orquantitation of T. vaginalis in a test sample.

A. DEFINITIONS

The following terms have the indicated meanings in the specificationunless expressly indicated to have a different meaning.

By “sample” or “test sample” is meant any substance suspected ofcontaining a target organism or nucleic acid derived from the targetorganism. The substance may be, for example, an unprocessed clinicalspecimen, such as a genitourinary tract specimen, a buffered mediumcontaining the specimen, a medium containing the specimen and lyticagents for releasing nucleic acid belonging to the target organism, or amedium containing nucleic acid derived from the target organism whichhas been isolated and/or purified in a reaction receptacle or on areaction material or device. In the claims, the terms “sample” and “testsample” may refer to specimen in its raw form or to any stage ofprocessing to release, isolate and purify nucleic acid derived fromtarget organisms in the specimen. Thus, within a method of use claim,each reference to a “sample” or “test sample” may refer to a substancesuspected of containing nucleic acid derived from the target organism ororganisms at different stages of processing and is not limited to theinitial form of the substance in the claim.

By “target nucleic acid” or “target” is meant a nucleic acid containinga target nucleic acid sequence.

By “target nucleic acid sequence,” “target sequence” or “target region”is meant a specific deoxyribonucleotide or ribonucleotide sequencecomprising all or part of the nucleotide sequence of a single-strandednucleic acid molecule.

By “oligonucleotide” or “oligomer” is meant a polymer made up of two ormore nucleoside subunits or nucleobase subunits coupled together. Theoligonucleotide may be DNA and/or RNA and analogs thereof. The sugargroups of the nucleoside subunits may be ribose, deoxyribose and analogsthereof, including, for example, ribonucleosides having a2′-O-methylsubstitution to the ribofuranosyl moiety. (Oligonucleotidesincluding nucleoside subunits having 2′ substitutions and which areuseful as detection probes, capture probes, and/or amplificationoligonucleotides are disclosed by Becker et al., “Method for AmplifyingTarget Nucleic Acids Using Modified Primers,” U.S. Pat. No. 6,130,038.)The nucleoside subunits may be joined by linkages such as phosphodiesterlinkages, modified linkages, or by non-nucleotide moieties which do notprevent hybridization of the oligonucleotide to its complementary targetnucleic acid sequence. Modified linkages include those linkages in whicha standard phosphodiester linkage is replaced with a different linkage,such as a phosphorothioate linkage or a methylphosphonate linkage. Thenucleobase subunits may be joined, for example, by replacing the naturaldeoxyribose phosphate backbone of DNA with a pseudo-peptide backbone,such as a 2-aminoethylglycine backbone which couples the nucleobasesubunits by means of a carboxymethyl linker to the central secondaryamine. (DNA analogs having a pseudo-peptide backbone are commonlyreferred to as “peptide nucleic acids” or “PNA”, and are disclosed byNielsen et al., “Peptide Nucleic Acids,” U.S. Pat. No. 5,539,082.) Othernon-limiting examples of oligonucleotides or oligomers contemplated bythe present disclosure include nucleic acid analogs containing bicyclicand tricyclic nucleoside and nucleotide analogs referred to as “LockedNucleic Acids,” “Locked Nucleoside Analogues” or “LNA.” (Locked NucleicAcids are disclosed by Wang, “Conformationally Locked Nucleosides andOligonucleotides,” U.S. Pat. No. 6,083,482; Imanishi et al.,“Bicyclonucleoside and Oligonucleotide Analogues,” U.S. Pat. No.6,268,490; and Wengel et al., “Oligonucleotide Analogues,” U.S. Pat. No.6,670,461.) Any nucleic acid analog is contemplated by the presentdisclosure, provided that the modified oligonucleotide can hybridize toa target nucleic acid under stringent hybridization conditions oramplification conditions. In the case of detection probes, the modifiedoligonucleotides must also be capable of preferentially hybridizing tothe target nucleic acid under stringent hybridization conditions.

Oligonucleotides of a defined sequence may be produced by techniquesknown to those of ordinary skill in the art, such as by chemical orbiochemical synthesis, and by in vitro or in vivo expression fromrecombinant nucleic acid molecules, e.g., bacterial or retroviralvectors. As intended by this disclosure, an oligonucleotide does notconsist of wild-type chromosomal DNA or the in vivo transcriptionproducts thereof. One use of an oligonucleotide is as a detection probe.Oligonucleotides may also be used as capture probes and amplificationoligonucleotides.

By “detection probe” or “probe” is meant a structure comprising anoligonucleotide having a base sequence sufficiently complementary to itstarget nucleic acid sequence to form a probe:target hybrid stable fordetection under stringent hybridization conditions. As would beunderstood by someone having ordinary skill in the art, theoligonucleotide is an isolated nucleic acid molecule, or an analogthereof, in a form not found in nature without human intervention (e.g.,recombined with foreign nucleic acid, isolated, or purified to someextent). The probes of this disclosure may have additional nucleosidesor nucleobases complementary to nucleotides outside of the targetedregion so long as such nucleosides or nucleobases do not preventhybridization under stringent hybridization conditions and, in the caseof detection probes, do not prevent preferential hybridization to thetarget nucleic acid. A non-complementary sequence may also be included,such as a target capture sequence (generally a homopolymer tract, suchas a poly-A, poly-T or poly-U tail), promotor sequence, a binding sitefor RNA transcription, a restriction endonuclease recognition site, orsequences which will confer a desired secondary or tertiary structure,such as a catalytic active site or a hairpin structure, which can beused to facilitate detection and/or amplification. Probes of a definedsequence may be produced by techniques known to those of ordinary skillin the art, such as by chemical synthesis, and by in vitro or in vivoexpression from recombinant nucleic acid molecules.

By “stable” or “stable for detection” is meant that the temperature of areaction mixture is at least 2° C. below the melting temperature of anucleic acid duplex. The temperature of the reaction mixture is more atleast 5° C. below the melting temperature of the nucleic acid duplex inone embodiment, or even more at least 10° C. below the meltingtemperature of the reaction mixture in another embodiment.

By “RNA and DNA equivalents” is meant RNA and DNA molecules having thesame complementary base pair hybridization properties. RNA and DNAequivalents have different sugar moieties (i.e., ribose versusdeoxyribose) and may differ by the presence of uracil in RNA and thyminein DNA. The differences between RNA and DNA equivalents do notcontribute to differences in homology because the equivalents have thesame degree of complementarity to a particular sequence.

By “hybridization” or “hybridize” is meant the ability of two completelyor partially complementary nucleic acid strands to come together underspecified hybridization assay conditions in a parallel or antiparallelorientation to form a stable structure having a double-stranded region.The two constituent strands of this double-stranded structure, sometimescalled a hybrid, are held together by hydrogen bonds. Although thesehydrogen bonds most commonly form between nucleotides containing thebases adenine and thymine or uracil (A and T or U) or cytosine andguanine (C and G) on single nucleic acid strands, base pairing can alsoform between bases which are not members of these “canonical” pairs.Non-canonical base pairing is well-known in the art. (See, e.g., RogerL. P. Adams et al., The Biochemistry of the Nucleic Acids (11^(th) ed.1992).)

By “preferentially hybridize” is meant that under stringenthybridization conditions, detection probes can hybridize to their targetnucleic acids to form stable probe:target hybrids indicating thepresence of at least one organism of interest, and there is not formed asufficient number of stable probe:non-target hybrids to indicate thepresence of non-targeted organisms, especially phylogenetically closelyrelated organisms. Thus, the probe hybridizes to target nucleic acid toa sufficiently greater extent than to non-target nucleic acid to enableone having ordinary skill in the art to accurately detect the presence(or absence) of nucleic acid derived from T. vaginalis, as appropriate,and distinguish its presence from that of a phylogenetically closelyrelated organism in a test sample. In general, reducing the degree ofcomplementarity between an oligonucleotide sequence and its targetsequence will decrease the degree or rate of hybridization of theoligonucleotide to its target region. However, the inclusion of one ormore non-complementary nucleosides or nucleobases may facilitate theability of an oligonucleotide to discriminate against non-targetorganisms.

Preferential hybridization can be measured using techniques known in theart and described herein, such as in the examples provided below. Someembodiments of preferential hybridization include when there is at leasta 10-fold difference between target and non-target hybridization signalsin a test sample, or when there is at least a 100-fold difference, orwhen there is at least a 1,000-fold difference. In general, non-targethybridization signals in a test sample are no more than the backgroundsignal level.

By “stringent hybridization conditions” or “stringent conditions” ismeant conditions permitting a detection probe to preferentiallyhybridize to a target nucleic acid (for example, rRNA or rDNA derivedfrom T. vaginalis) and not to nucleic acid derived from a closelyrelated non-target microorganism. Stringent hybridization conditions mayvary depending upon factors including the GC content and length of theprobe, the degree of similarity between the probe sequence and sequencesof non-target sequences which may be present in the test sample, and thetarget sequence. Hybridization conditions include the temperature andthe composition of the hybridization reagents or solutions. Specifichybridization assay conditions are set forth infra in the Examplessection and in the section entitled “Detection Probes to Trichomonasvaginalis Ribosomal Nucleic Acid.” Exemplary hybridization conditionsfor detecting target nucleic acids derived from T. vaginalis with theprobes of the present disclosure include a temperature of about 60° C.and a salt concentration of about 1.5 M. Other acceptable stringenthybridization conditions could be easily ascertained by someone havingordinary skill in the art.

By “assay conditions” is meant conditions permitting stablehybridization of an oligonucleotide to a target nucleic acid. Assayconditions do not require preferential hybridization of theoligonucleotide to the target nucleic acid.

By “nucleic acid duplex,” “duplex,” “nucleic acid hybrid” or “hybrid” ismeant a stable nucleic acid structure comprising a double-stranded,hydrogen-bonded region. Such hybrids include RNA:RNA, RNA:DNA andDNA:DNA duplex molecules and analogs thereof. The structure issufficiently stable to be detectable by any known means, including meansthat do not require a probe associated label. For instance, thedetection method may include a probe-coated substrate that is opticallyactive and sensitive to changes in mass at its surface. Mass changesresult in different reflective and transmissive properties of theoptically active substrate in response to light and serve to indicatethe presence or amount of immobilized target nucleic acid. (Thisexemplary form of optical detection is disclosed by Nygren et al.,“Devices and Methods for Optical Detection of Nucleic AcidHybridization,” U.S. Pat. No. 6,060,237.) Other means for detecting theformation of a nucleic acid duplex that do not require the use of alabeled probe include the use of binding agents, which includeintercalating agents such as ethidium bromide. See, e.g., Higuchi,“Homogenous Methods for Nucleic Amplification and Detection,” U.S. Pat.No. 5,994,056.

By “amplification oligonucleotide” or “primer” is meant anoligonucleotide capable of hybridizing to a target nucleic acid andacting as a primer and/or a promoter template (e.g., for synthesis of acomplementary strand, thereby forming a functional promoter sequence)for the initiation of nucleic acid synthesis. If the amplificationoligonucleotide is designed to initiate RNA synthesis, the primer maycontain a base sequence which is non-complementary to the targetsequence but which is recognized by a RNA polymerase such as a T7, T3,or SP6 RNA polymerase. An amplification oligonucleotide may contain a 3′terminus that is modified to prevent or lessen the rate or amount ofprimer extension. (McDonough et al., “Methods of Amplifying NucleicAcids Using Promoter-Containing Primer Sequences,” U.S. Pat. No.5,766,849, disclose primers and promoter-primers having modified orblocked 3′-ends.) While the amplification oligonucleotides of thepresent disclosure may be chemically synthesized or derived from avector, they are not naturally occurring nucleic acid molecules.

By “nucleic acid amplification” or “target amplification” is meantincreasing the number of nucleic acid molecules having at least onetarget nucleic acid sequence. Target amplification according to thepresent disclosure may be either linear or exponential.

By “amplification conditions” is meant conditions permitting nucleicacid amplification. Acceptable amplification conditions could be readilyascertained without the exercise of anything more than routineexperimentation by someone having ordinary skill in the art depending onthe particular method of amplification employed.

By “antisense,” “opposite sense,” or “negative sense” is meant a nucleicacid molecule perfectly complementary to a reference, or sense, nucleicacid molecule.

By “sense,” “same-sense,” or “positive sense” is meant a nucleic acidmolecule perfectly homologous to a reference nucleic acid molecule.

By “amplicon” is meant a nucleic acid molecule generated in a nucleicacid amplification reaction and which is derived from a target nucleicacid. An amplicon contains a target nucleic acid sequence that may be ofthe same or opposite sense as the target nucleic acid.

By “derived” is meant that the referred to nucleic acid is obtaineddirectly from an organism or is the product of a nucleic acidamplification. Thus, a nucleic acid that is “derived” from an organismmay be, for example, an antisense RNA molecule which does not naturallyexist in the organism.

By “capture probe” is meant an oligonucleotide that is capable ofbinding to a target nucleic acid (generally in a region other than thattargeted by a detection probe) and, either directly or indirectly, to asolid support, thereby providing means for immobilizing and isolatingthe target nucleic acid in a test sample. The capture probe includes atarget-complementary base sequence that hybridizes to the target nucleicacid. In one embodiment the capture probes may include a member ofligand-ligate binding pair (e.g., avidin-biotin linkage) forimmobilizing the capture probe on a solid support. In another embodimentthe capture probes include an immobilized probe binding region thathybridizes to an immobilized probe bound to a solid support. While thecapture probe hybridizes to both the target nucleic acid and theimmobilized probe under stringent conditions, the target-complementarybase sequence and the immobilized probe binding regions of the captureprobe may be designed to bind to their target sequences under differenthybridization conditions. In this way, the capture probe may be designedso that it first hybridizes to the target nucleic acid under morefavorable in solution kinetics before adjusting the conditions to permithybridization of the immobilized probe binding region to the immobilizedprobe. The target-complementary base sequence and immobilized probebinding regions may be contained within the same oligonucleotide,directly adjoining each other or separated by one or more optionallymodified nucleotides, or these regions may be joined to each other bymeans of a non-nucleotide linker.

By “target-complementary base sequence” is meant that portion of anoligonucleotide which stably binds to a target sequence present in atarget nucleic acid, or the complement of the target sequence, underassay conditions. The assay conditions may be stringent hybridizationconditions or amplification conditions.

By “non-complementary” is meant that portion of an oligonucleotide whichdoes not stably bind to a target sequence present in a target nucleicacid, or the complement of the target sequence, under assay conditions.The assay conditions may be stringent hybridization conditions oramplification conditions.

By “immobilized probe binding region” is meant that portion of anoligonucleotide which hybridizes to an immobilized probe under assayconditions.

By “homopolymer” tail in the claims is meant a contiguous base sequenceof at least 10 identical bases (e.g., 10 contiguous adenines orthymines).

By “immobilized probe” is meant an oligonucleotide for joining a captureprobe to an immobilized support. The immobilized probe is joined eitherdirectly or indirectly to the solid support by a linkage or interactionwhich remains stable under the conditions employed to hybridize thecapture probe to the target nucleic acid and to the immobilized probe,whether those conditions are the same or different. The immobilizedprobe facilitates separation of the bound target nucleic acid fromunbound materials in a sample.

By “isolate” or “isolating” is meant that at least a portion of thetarget nucleic acid present in a test sample is concentrated within areaction receptacle or on a reaction device or solid carrier (e.g., testtube, cuvette, microtiter plate well, nitrocellulose filter, slide orpipette tip) in a fixed or releasable manner so that the target nucleicacid can be purified without significant loss of the target nucleic acidfrom the receptacle, device or carrier.

By “purify” or “purifying” is meant that one or more components of thetest sample are removed from one or more other components of the sample.Sample components to be purified may include viruses, nucleic acids or,in particular, target nucleic acids in a generally aqueous solutionphase which may also include undesirable materials such as proteins,carbohydrates, lipids, non-target nucleic acid and/or labeled probes. Insome embodiments, the purifying step removes at least about 70%, or atleast about 90%, or at least about 95% of the undesirable componentspresent in the sample.

By “phylogenetically closely related” is meant that the organisms areclosely related to each other in an evolutionary sense and thereforewould be expected to have a higher total nucleic acid sequence homologythan organisms that are more distantly related. Organisms occupyingadjacent and next to adjacent positions on the phylogenetic tree areclosely related. Organisms occupying positions farther away thanadjacent or next to adjacent positions on the phylogenetic tree willstill be closely related if they have significant total nucleic acidsequence homology.

B. HYBRIDIZATION CONDITIONS AND PROBE DESIGN

Hybridization reaction conditions, most importantly the temperature ofhybridization and the concentration of salt in the hybridizationsolution, can be selected to allow the detection probes or, in somecases, amplification oligonucleotides of the present disclosure topreferentially hybridize to a T. vaginalis-derived target nucleic acidand not to other non-target nucleic acids suspected of being present ina test sample. At decreased salt concentrations and/or increasedtemperatures (conditions of increased stringency) the extent of nucleicacid hybridization decreases as hydrogen bonding between pairednucleobases in the double-stranded hybrid molecule is disrupted. Thisprocess is known as “melting.”

Generally speaking, the most stable hybrids are those having the largestnumber of contiguous, perfectly matched (i.e., hydrogen-bonded)nucleotide base pairs. Such hybrids would usually be expected to be thelast to melt as the stringency of the hybridization conditionsincreases. However, a double-stranded nucleic acid region containing oneor more mismatched, “non-canonical,” or imperfect base pairs (resultingin weaker or non-existent base pairing at that position in thenucleotide sequence of a nucleic acid) may still be sufficiently stableunder conditions of relatively high stringency to allow the nucleic acidhybrid to be formed and detected in a hybridization assay withoutcross-reacting with other, non-selected nucleic acids which may bepresent in a test sample.

Hence, depending on the degree of similarity between the nucleotidesequences of the target nucleic acid and those of non-target nucleicacids belonging to phylogenetically distinct, but closely-relatedorganisms on one hand, and the degree of complementarity between thenucleotide sequences of a particular detection probe or amplificationoligonucleotide and those of the target and non-target nucleic acids onthe other, one or more mismatches will not necessarily defeat theability of an oligonucleotide contained in the probe or amplificationoligonucleotide to hybridize to the target nucleic acid and not tonon-target nucleic acids.

The detection probes of the present disclosure were chosen, selected,and/or designed to maximize the difference between the meltingtemperatures of the probe:target hybrid (T_(m), defined as thetemperature at which half of the potentially double-stranded moleculesin a given reaction mixture are in a single-stranded, denatured state)and the T_(m) of a mismatched hybrid formed between the probe andribosomal RNA (rRNA) or ribosomal DNA (rDNA) of the phylogeneticallymost closely-related organisms expected to be present in the testsample, but not sought to be detected. While the unlabeled amplificationoligonucleotides and capture probes need not have such an extremely highdegree of specificity as the detection probe to be useful in the presentdisclosure, they are designed in a similar manner to preferentiallyhybridize to one or more target nucleic acids over other nucleic acidsunder specified amplification, assay or stringent hybridizationconditions.

To facilitate the identification of nucleic acid sequences to be used inthe design of probes, nucleotide sequences from different organisms werefirst aligned to maximize homology. The source organisms and theassociated nucleotide sequences used for this comparison were obtainedfrom the GenBank database and had the following accession numbers:Trichomonas vaginalis (Accession No. U17510), Trimastix pyriformis(Accession No. AF244903), Dientamoeba fragilis (Accession No. U37461),Trichomonas gallinae (Accession No. U86614), Trichomonas tenax(Accession Nos. D49495 and U37711), Tetratrichomonas gallinarum(Accession No. AF124608), Kalotermes flavicollis (Accession No.AF215856), Trichomitus trypanoides (Accession No. X79559), Hodotermopsissjoestedti (Accession No. AB032234), Pentatrichomonas hominis (AccessionNo. AF124609), Pseudotrypanosoma giganteum (Accession No. AF052706),Ditrichomonas honigbergi (Accession No. U17505), Monotrichomonas speciesATCC No. 50693 (Accession No. AF072905), Pseudotrichomonas keilini(Accession No. U17511), Monocercomonas species ATCC No. 50210 (AccessionNo. U17507), Tritrichomonas foetus (Accession No. U17509) and Entamoebahistolytica (Accession No. X64142).

Within the rRNA molecule there is a close relationship between secondarystructure (caused in part by intra-molecular hydrogen bonding) andfunction. This fact imposes restrictions on evolutionary changes in theprimary nucleotide sequence causing the secondary structure to bemaintained. For example, if a base is changed in one “strand” of adouble helix (due to intra-molecular hydrogen bonding, both “strands”are part of the same rRNA molecule), a compensating substitution usuallyoccurs in the primary sequence of the other “strand” in order topreserve complementarity (this is referred to as co-variance), and thusthe necessary secondary structure. This allows two very different rRNAsequences to be aligned based both on the conserved primary sequence andalso on the conserved secondary structure elements. Potential targetsequences for the detection probes described herein were identified bynoting variations in the homology of the aligned sequences.

The sequence evolution at each of the variable regions is mostlydivergent. Because of the divergence, corresponding rRNA variableregions of more distant phylogenetic relatives of T. vaginalis showgreater differences from T. vaginalis rRNA than do the rRNAs ofphylogenetically closer relatives. Sufficient variation between T.vaginalis and other organisms was observed to identify potential targetsites and to design detection probes useful for distinguishing T.vaginalis over non-T. vaginalis organisms in a test sample, particularlyTrichomonas tenax, the most closely related organism to T. vaginalis.

Merely identifying putatively unique potential target nucleotidesequences does not guarantee that a functionally species-specificdetection probe may be made to hybridize to T. vaginalis rRNA or rDNAcomprising that sequence. Various other factors will determine thesuitability of a nucleic acid locus as a target site for genus-specificor species-specific probes. Because the extent and specificity ofhybridization reactions such as those described herein are affected by anumber of factors, manipulation of one or more of those factors willdetermine the exact sensitivity and specificity of a particularoligonucleotide, whether perfectly complementary to its target or not.The importance and effect of various assay conditions are known to thoseskilled in the art and are disclosed by Hogan et al., “Nucleic AcidProbes for Detection and/or Quantitation of Non-Viral Organisms,” U.S.Pat. No. 5,840,488; Hogan et al., “Nucleic Acid Probes to Mycobacteriumgordonae,” U.S. Pat. No. 5,216,143; and Kohne, “Method for Detection,Identification and Quantitation of Non-Viral Organisms,” U.S. Pat. No.4,851,330. The contents of each of the foregoing references is herebyincorporated by reference herein.

The desired temperature of hybridization and the hybridization solutioncomposition (such as salt concentration, detergents, and other solutes)can also greatly affect the stability of double-stranded hybrids.Conditions such as ionic strength and the temperature at which a probewill be allowed to hybridize to a target must be taken into account inconstructing a genus-specific or species-specific probe. The thermalstability of hybrid nucleic acids generally increases with the ionicstrength of the reaction mixture. On the other hand, chemical reagentsthat disrupt hydrogen bonds, such as formamide, urea, dimethyl sulfoxideand alcohols, can greatly reduce the thermal stability of the hybrids.

To maximize the specificity of a probe for its target, the subjectprobes of the present disclosure were designed to hybridize to theirtargets under conditions of high stringency. Under such conditions onlysingle nucleic acid strands having a high degree of complementarity willhybridize to each other. Single nucleic acid strands without such a highdegree of complementarity will not form hybrids. Accordingly, thestringency of the assay conditions determines the amount ofcomplementarity that should exist between two nucleic acid strands inorder to form a hybrid. Stringency is chosen to maximize the differencein stability between the hybrid formed between the probe and the targetnucleic acid and potential hybrids between the probe and any non-targetnucleic acids present in a test sample.

Proper specificity may be achieved by minimizing the length of thedetection probe having perfect complementarity to sequences ofnon-target organisms, by avoiding G and C rich regions ofcomplementarity to non-target nucleic acids, and by constructing theprobe to contain as many destabilizing mismatches to non-targetsequences as possible. Whether a probe is appropriate for detecting onlya specific type of organism depends largely on the thermal stabilitydifference between probe:target hybrids versus probe:non-target hybrids.In designing probes, the differences in these T_(m) values should be aslarge as possible (for example, 2-5° C. or more). Manipulation of theT_(m) can be accomplished by changes to probe length and probecomposition (e.g., GC content versus AT content).

In general, the optimal hybridization temperature for oligonucleotideprobes is approximately 5° C. below the melting temperature for a givenduplex. Incubation at temperatures below the optimum temperature mayallow mismatched base sequences to hybridize and can therefore decreasespecificity. The longer the probe, the more hydrogen bonding betweenbase pairs and, in general, the higher the T_(m). Increasing thepercentage of G and C also increases the T_(m) because G-C base pairsexhibit additional hydrogen bonding and therefore greater thermalstability than A-T base pairs. Such considerations are known in the art.(See, e.g., J. Sambrook et al., Molecular Cloning: A Laboratory Manual,ch. 11 (2^(nd) ed. 1989).)

One method to determine T_(m) measures hybridization using the wellknown Hybridization Protection Assay (HPA) disclosed by Arnold et al.,“Homogenous Protection Assay,” U.S. Pat. No. 5,283,174, the contents ofwhich are hereby incorporated by reference herein. The T_(m) can bemeasured using HPA in the following manner. Probe molecules are labeledwith an acridinium ester and permitted to form probe:target hybrids in alithium succinate buffer (0.1 M lithium succinate buffer, pH 4.7, 20 mMEDTA, 15 mM aldrithiol-2, 1.2 M LiCl, 3% (v/v) ethanol absolute, 2%(w/v) lithium lauryl sulfate) using an excess amount of target. Aliquotsof the solution containing the probe:target hybrids are then diluted inthe lithium succinate buffered solution and incubated for five minutesat various temperatures starting below that of the anticipated T_(m)(typically 55° C.) and increasing in 2-5° C. increments. This solutionis then diluted with a mild alkaline borate buffer (600 mM boric acid,240 mM NaOH, 1% (v/v) TRITON® X-100 detergent, pH 8.5) and incubated atan equal or lower temperature (for example 50° C.) for ten minutes.

Under these conditions the acridinium ester attached to thesingle-stranded probe is hydrolyzed, while the acridinium ester attachedto hybridized probe is relatively protected from hydrolysis. Thus, theamount of acridinium ester remaining after hydrolysis treatment isproportional to the number of hybrid molecules. The remaining acridiniumester can be measured by monitoring the chemiluminescence produced fromthe remaining acridinium ester by adding hydrogen peroxide and alkali tothe solution. Chemiluminescence can be measured in a luminometer, suchas a LEADER® Luminometer (Gen-Probe Incorporated; San Diego, Calif.).The resulting data is plotted as percent of maximum signal (usually fromthe lowest temperature) versus temperature. The T_(m) is defined as thetemperature at which 50% of the maximum signal remains. In addition tothe method above, T_(m) may be determined by isotopic methods known tothose skilled in the art (see, e.g., Hogan et al., U.S. Pat. No.5,840,488).

To ensure specificity of a detection probe for its target, it ispreferable to design probes that hybridize only to target nucleic acidunder conditions of high stringency. Only highly complementary sequenceswill form hybrids under conditions of high stringency. Accordingly, thestringency of the assay conditions determines the amount ofcomplementarity needed between two sequences in order for a stablehybrid to form. Stringency should be chosen to maximize the differencein stability between the probe:target hybrid and potentialprobe:non-target hybrids.

Examples of specific stringent hybridization conditions are provided inthe Examples section infra. Of course, alternative stringenthybridization conditions can be determined by those of ordinary skill inthe art based on the present disclosure. (See, e.g., Sambrook et al.,supra, ch. 11.)

The length of the target nucleic acid sequence region and, accordingly,the length of the probe sequence can also be important. In some cases,there may be several sequences from a particular region, varying inlocation and length, which may be used to design probes with the desiredhybridization characteristics. In other cases, one probe may besignificantly better with regard to specificity than another thatdiffers from it merely by a single base. While it is possible fornucleic acids that are not perfectly complementary to hybridize, thelongest stretch of perfectly complementary bases, as well as the basecompositions, will generally determine hybrid stability.

Regions of rRNA known to form strong internal structures inhibitory tohybridization are less preferred target regions. Likewise, probes withextensive self-complementarity are generally to be avoided, withspecific exceptions being discussed below. If a strand is wholly orpartially involved in an intramolecular or intermolecular hybrid, itwill be less able to participate in the formation of a newintermolecular probe:target hybrid without a change in the reactionconditions. Ribosomal RNA molecules are known to form very stableintramolecular helices and secondary structures by hydrogen bonding. Bydesigning a probe to a region of the target nucleic acid which remainssubstantially single-stranded under hybridization conditions, the rateand extent of hybridization between probe and target may be increased.

A genomic ribosomal nucleic acid (rDNA) target occurs naturally in adouble-stranded form, as does the product of the polymerase chainreaction (PCR). These double-stranded targets are naturally inhibitoryto hybridization with a probe and require denaturation prior tohybridization. Appropriate denaturation and hybridization conditions areknown in the art (see, e.g., Southern, E. M., J. Mol. Biol., 98:503(1975)).

A number of formulae are available which will provide an estimate of themelting temperature for perfectly matched oligonucleotides to theirtarget nucleic acids. One such formula is the following:T_(m)=81.5+16.6(log₁₀[Na+])+0.41(fraction G+C)−(600/N) (where N=thelength of the oligonucleotide in number of nucleotides) provides a goodestimate of the T_(m) for oligonucleotides between 14 and 60 to 70nucleotides in length. From such calculations, subsequent empiricalverification or “fine tuning” of the T_(m) may be made using screeningtechniques well known in the art. For further information onhybridization and oligonucleotide probes reference may be made toSambrook et al., supra, ch. 11. This reference, among others well knownin the art, also provides estimates of the effect of mismatches on theT_(m) of a hybrid. Thus, from the known nucleotide sequence of a givenregion of the ribosomal RNA (or rDNA) of two or more organisms,oligonucleotides may be designed which will distinguish these organismsfrom one another.

C. NUCLEIC ACID AMPLIFICATION

Amplification oligonucleotides of the present disclosure may beoligodeoxynucleotides and are sufficiently long to be used as asubstrate for the synthesis of extension products by a nucleic acidpolymerase. Optimal amplification oligonucleotide length should takeinto account several factors, including the temperature of reaction, thestructure and base composition of the amplification oligonucleotide, andhow the amplification oligonucleotide is to be used. For example, foroptimal specificity the oligonucleotide amplification oligonucleotidegenerally should be at least 12 bases in length, depending on thecomplexity of the target nucleic acid sequence. If such specificity isnot essential, shorter amplification oligonucleotides may be used. Insuch a case, it may be desirable to carry out the reaction at coolertemperatures in order to form stable hybrid complexes with the templatenucleic acid.

Useful guidelines for designing amplification oligonucleotides anddetection probes with desired characteristics are described infra in thesection entitled “Preparation of Oligonucleotides.” Optimal sites foramplifying and probing contain at least two or three conserved regionsof T. vaginalis nucleic acid. These regions are about 15 to 350 bases inlength in one embodiment, or between about 15 and 150 bases in length inanother embodiment.

The degree of amplification observed with a set of amplificationoligonucleotides (e.g., primers and/or promoter-primers) depends onseveral factors, including the ability of the amplificationoligonucleotides to hybridize to their specific target sequences andtheir ability to be extended or copied enzymatically. Whileamplification oligonucleotides of different lengths and basecompositions may be used, amplification oligonucleotides in thisdisclosure have target-complementary base sequences of 18 to 40 baseswith a predicted T_(m) to target of about 42° C.

Parameters affecting probe hybridization, such as T_(m),complementarity, and secondary structure of the target sequence, alsoaffect amplification oligonucleotide hybridization and thereforeperformance of the amplification oligonucleotides. The degree ofnon-specific extension (primer-dimer or non-target copying) can alsoaffect amplification efficiency. Thus, amplification oligonucleotidesare selected to have low self-complementarity or cross-complementarity,particularly at the 3′ ends of their sequences. Notwithstanding, itshould be noted that the “signal primers” described infra could bemodified to include regions of self-complementarity, therebytransforming them into “molecular torch” or “molecular beacon” signalprimers, such as these terms are defined below. Lengthy homopolymer runsand high GC content are avoided to reduce spurious primer extension.Computer programs are available to aid in this aspect of the design,including Oligo Tech analysis software which is available from OligosEtc. Inc. and can be accessed on the World Wide Web at the followingURL: http://www.oligosetc.com.

A nucleic acid polymerase used in conjunction with the amplificationoligonucleotides of the present disclosure refers to a chemical,physical, or biological agent that incorporates either ribonucleotidesor deoxyribonucleotides, or both, into a nucleic acid polymer, orstrand, in a template-dependent manner. Examples of nucleic acidpolymerases include DNA-directed DNA polymerases, RNA-directed DNApolymerases, and RNA-directed RNA polymerases. DNA polymerases bringabout nucleic acid synthesis in a template-dependent manner and in a 5′to 3′ direction. Because of the typical anti-parallel orientation of thetwo strands in a double-stranded nucleic acid, this direction is from a3′ region on the template to a 5′ region on the template. Examples ofDNA-directed DNA polymerases include E. coli DNA polymerase I, thethermostable DNA polymerase from Thermus aquaticus (Taq), and the largefragment of DNA polymerase I from Bacillus stearothermophilis (Bst).Examples of RNA directed DNA polymerases include various retroviralreverse transcriptases, such as Moloney murine leukemia virus (MMLV)reverse transcriptase or avian myeloblastosis virus (AMV) reversetranscriptase.

During most nucleic acid amplification reactions, a nucleic acidpolymerase adds nucleotide residues to the 3′ end of the primer usingthe target nucleic acid as a template, thus synthesizing a secondnucleic acid strand having a nucleotide sequence partially or completelycomplementary to a region of the target nucleic acid. In many nucleicacid amplification reactions, the two strands comprising the resultingdouble-stranded structure must be separated by chemical or physicalmeans in order to allow the amplification reaction to proceed.Alternatively, the newly synthesized template strand may be madeavailable for hybridization with a second primer or promoter-primer byother means, such as through strand displacement or the use of anucleolytic enzyme which digests part or all of the original targetstrand. In this way the process may be repeated through a number ofcycles, resulting in a large increase in the number of nucleic acidmolecules having the target nucleotide sequence.

Either the first or second amplification oligonucleotide, or both, maybe a promoter-primer. (In some applications, the amplificationoligonucleotides may only consist of promoter-primers which arecomplementary to the sense strand, as disclosed by Kacian et al.,“Nucleic Acid Sequence Amplification Method, Composition and Kit,” U.S.Pat. No. 5,554,516.) A promoter-primer usually contains anoligonucleotide that is not complementary to a nucleotide sequencepresent in the target nucleic acid molecule or primer extensionproduct(s) (see Kacian et al., “Nucleic Acid Sequence AmplificationMethods,” U.S. Pat. No. 5,399,491, for a description of sucholigonucleotides). These non-complementary sequences may be located 5′to the complementary sequences on the amplification oligonucleotide andmay provide a locus for initiation of RNA synthesis when madedouble-stranded through the action of a nucleic acid polymerase. Thepromoter thus provided may allow for the in vitro transcription ofmultiple RNA copies of the target nucleic acid sequence. It will beappreciated that when reference is made to a primer in thisspecification, such reference is intended to include the primer aspectof a promoter-primer as well, unless the context of the referenceclearly indicates otherwise.

In some amplification systems (see, e.g., the amplification methodsdisclosed by Dattagupta et al., “Isothermal Strand DisplacementAmplification,” U.S. Pat. No. 6,087,133), the amplificationoligonucleotides may contain 5′ non-complementary nucleotides whichassist in strand displacement. Furthermore, when used in conjunctionwith a nucleic acid polymerase having 5′ exonuclease activity, theamplification oligonucleotides may have modifications at their 5′ end toprevent enzymatic digestion. Alternatively, the nucleic acid polymerasemay be modified to remove the 5′ exonuclease activity, such as bytreatment with a protease that generates an active polymerase fragmentwith no such nuclease activity. In such a case the primers need not bemodified at their 5′ ends.

1. Preparation of Oligonucleotides

The detection probes, capture probes, and amplification oligonucleotidesof the present disclosure can be readily prepared by methods known inthe art. In one embodiment, the oligonucleotides are synthesized usingsolid phase methods. For example, Caruthers describes using standardphosphoramidite solid-phase chemistry to join nucleotides byphosphodiester linkages. See Caruthers et al., “Chemical Synthesis ofDeoxynucleotides by the Phosphoramidite Method,” Methods Enzymol.,154:287 (1987). Automated solid-phase chemical synthesis usingcyanoethyl phosphoramidite precursors has been described by Barone. SeeBarone et al., “In Situ Activation of bis-dialkylaminephosphines—a NewMethod for Synthesizing Deoxyoligonucleotides on Polymer Supports,”Nucleic Acids Res., 12(10):4051 (1984). Likewise, Batt, “Method andReagent for Sulfurization of Organophosphorous Compounds,” U.S. Pat. No.5,449,769, discloses a procedure for synthesizing oligonucleotidescontaining phosphorothioate linkages. In addition, Riley et al.,“Process for the Purification of Oligomers,” U.S. Pat. No. 5,811,538disclose the synthesis of oligonucleotides having different linkages,including methylphosphonate linkages. Moreover, methods for the organicsynthesis of oligonucleotides are known to those of skill in the art andare described in, for example, Sambrook et al., supra, ch. 10.

Following synthesis of a particular oligonucleotide, several differentprocedures may be utilized to purify and control the quality of theoligonucleotide. Suitable procedures include polyacrylamide gelelectrophoresis or high pressure liquid chromatography. Both of theseprocedures are well known to those skilled in the art.

All of the oligonucleotides of the present disclosure, whether detectionprobes, capture probes or amplification oligonucleotides, may bemodified with chemical groups to enhance their performance or tofacilitate the characterization of amplification products.

For example, backbone-modified oligonucleotides such as those havingphosphorothioate, methylphosphonate, 2′-O-alkyl, or peptide groups whichrender the oligonucleotides resistant to the nucleolytic activity ofcertain polymerases or to nuclease enzymes may allow the use of suchenzymes in an amplification or other reaction. Another example of amodification involves using non-nucleotide linkers incorporated betweennucleotides in the nucleic acid chain of a probe or primer, and which donot prevent hybridization of a probe or hybridization and elongation ofa primer. (See Arnold et al., “Non-Nucleotide Linking Reagents forNucleotide Probes,” U.S. Pat. No. 6,031,091, the contents of which arehereby incorporated by reference herein.) The oligonucleotides of thepresent disclosure may also contain mixtures of the desired modified andnatural nucleotides.

The 3′ end of an amplification oligonucleotide, particularly apromoter-primer, may be modified or blocked to prevent or inhibitinitiation of DNA synthesis, as disclosed by Kacian et al., U.S. Pat.No. 5,554,516. The 3′ end of the primer can be modified in a variety ofways well known in the art. By way of example, appropriate modificationsto a promoter-primer can include the addition of ribonucleotides, 3′deoxynucleotide residues (e.g., cordycepin), 2′,3′-dideoxynucleotideresidues, modified nucleotides such as phosphorothioates, andnon-nucleotide linkages such as those disclosed by Arnold et al. in U.S.Pat. No. 6,031,091 or alkane-diol modifications (see Wilk et al.,“Backbone-Modified Oligonucleotides Containing a Butanediol-1,3 Moietyas a ‘Vicarious Segment’ for the Deoxyribosyl Moiety—Synthesis andEnzyme Studies,” Nucleic Acids Res., 18(8):2065 (1990)), or themodification may simply consist of a region 3′ to the priming sequencethat is non-complementary to the target nucleic acid sequence.Additionally, a mixture of different 3′ blocked promoter-primers or of3′ blocked and unblocked promoter-primers may increase the efficiency ofnucleic acid amplification, as described therein.

As disclosed above, the 5′ end of primers may be modified to beresistant to the 5′-exonuclease activity present in some nucleic acidpolymerases. Such modifications can be carried out by adding anon-nucleotide group to the terminal 5′ nucleotide of the primer usingtechniques such as those disclosed by Arnold et al., U.S. Pat. No.6,031,091.

Once synthesized, a selected oligonucleotide may be labeled by any wellknown method (see, e.g., Sambrook et al., supra, ch. 10). Useful labelsinclude radioisotopes as well as non-radioactive reporting groups.Isotopic labels include ³H, ³⁵S, ³²P, ¹²⁵I, ⁵⁷Co, and ¹⁴C. Isotopiclabels can be introduced into the oligonucleotide by techniques known inthe art such as nick translation, end labeling, second strand synthesis,the use of reverse transcription, and by chemical methods. When usingradiolabeled probes, hybridization can be detected by autoradiography,scintillation counting, or gamma counting. The detection method selectedwill depend upon the particular radioisotope used for labeling.

Non-isotopic materials can also be used for labeling and may beintroduced internally into the nucleic acid sequence or at the end ofthe nucleic acid sequence. Modified nucleotides may be incorporatedenzymatically or chemically. Chemical modifications of the probe may beperformed during or after synthesis of the probe, for example, throughthe use of non-nucleotide linker groups as disclosed by Arnold et al.,U.S. Pat. Nos. 5,585,481, 5,639,604, and 6,031,091. Non-isotopic labelsinclude fluorescent molecules (individual labels or combinations oflabels, such as the fluorescence resonance energy transfer (FRET) pairsdisclosed by Tyagi et al., “Detectably Labeled Dual ConformationOligonucleotide Probes,” U.S. Pat. No. 5,925,517), chemiluminescentmolecules, enzymes, cofactors, enzyme substrates, haptens, or otherligands.

In one embodiment, the detection probes of the present disclosure arelabeled using a non-nucleotide linker with an acridinium ester.Acridinium ester labeling may be performed as disclosed by Arnold etal., “Acridinium Ester Labelling and Purification of Nucleotide Probes,”U.S. Pat. No. 5,185,439, the contents of which are hereby incorporatedby reference herein.

2. Amplification of Trichomonas vaginalis Ribosomal Nucleic Acid

The amplification oligonucleotides of the present disclosure aredirected to 18S regions of ribosomal nucleic acid derived from T.vaginalis. These amplification oligonucleotides may flank, overlap, orbe contained within at least one of the target sequences of a detectionprobe (or its complement) used to detect the presence of T. vaginalis ina nucleic acid amplification assay. As indicated above, theamplification oligonucleotides may also include non-complementary basesat their 5′ ends comprising a promoter sequence able to bind a RNApolymerase and direct RNA transcription using the target nucleic acid asa template. A T7 promoter sequence, such as SEQ ID NO:54, may be used.Examples of amplification oligonucleotides are listed in Table 1

TABLE 1  T. vaginalis 18S amplification oligonucleotides SEQ ID NO.Sequence 45 gctaacgagcgagattatcgcc 46 gcuaacgagcgagauuaucgcc 47ggcgataatctcgctcgttagc 48 ggcgauaaucucgcucguuagc 49ggcatcacggacctgttattgc 50 gcaauaacagguccgugaugcc 51ggcatcacggacctgttattgc 52 ggcaucacggaccuguuauugc 53aatttaatacgactcactatagggagaggcatcacg gacctgttattgc

In one embodiment, a set of at least two amplification oligonucleotidesfor amplifying T. vaginalis-derived nucleic acid is provided whichincludes: (i) a first amplification oligonucleotide having a basesequence comprising, overlapping with, consisting essentially of,consisting of, substantially corresponding to, or contained within thebase sequence of SEQ ID Nos. 45-48; and (ii) a second amplificationoligonucleotide having a base sequence comprising, overlapping with,consisting essentially of, consisting of, substantially correspondingto, or contained within the base sequence of SEQ ID Nos. 49-52. Thesecond amplification oligonucleotide may include a 5′ promoter sequence(e.g., the T7 promoter sequence of SEQ ID NO:54) as shown in SEQ IDNO:53.

Amplification oligonucleotides of the present disclosure may havemodifications, such as blocked 3′ and/or 5′ termini (as discussed above)or sequence additions including, but not limited to, a specificnucleotide sequence recognized by a RNA polymerase (e.g., a promotersequence for T7, T3 or SP6 RNA polymerase), a sequence which enhancesinitiation or elongation of RNA transcription by a RNA polymerase, or asequence which may provide for intra-molecular base pairing andencourage the formation of secondary or tertiary nucleic acidstructures.

Amplification oligonucleotides are used in any suitable nucleic acidamplification procedure now known or later developed. Existingamplification procedures include the polymerase chain reaction (PCR),transcription-mediated amplification (TMA), nucleic acid sequence-basedamplification (NASBA), self-sustained sequence replication (3SR), ligasechain reaction (LCR), strand displacement amplification (SDA), andLoop-Mediated Isothermal Amplification (LAMP), each of which is wellknown in the art. See, e.g., Mullis, “Process for Amplifying NucleicAcid Sequences,” U.S. Pat. No. 4,683,202; Erlich et al., “Kits forAmplifying and Detecting Nucleic Acid Sequences,” U.S. Pat. No.6,197,563; Walker et al., Nucleic Acids Res., 20:1691-1696 (1992); Fahyet al., “Self-sustained Sequence Replication (3SR): An IsothermalTranscription-Based Amplification System Alternative to PCR,” PCRMethods and Applications, 1:25-33 (1991); Kacian et al., U.S. Pat. No.5,399,491; Kacian et al., “Nucleic Acid Sequence Amplification Methods,”U.S. Pat. No. 5,480,784; Davey et al., “Nucleic Acid AmplificationProcess,” U.S. Pat. No. 5,554,517; Birkenmeyer et al., “Amplification ofTarget Nucleic Acids Using Gap Filling Ligase Chain Reaction,” U.S. Pat.No. 5,427,930; Marshall et al., “Amplification of RNA Sequences Usingthe Ligase Chain Reaction,” U.S. Pat. No. 5,686,272; “Walker, StrandDisplacement Amplification,” U.S. Pat. No. 5,712,124; Notomi et al.,“Process for Synthesizing Nucleic Acid,” European Patent Application No.1 020 534 A1; Dattagupta et al., “Isothermal Strand DisplacementAmplification,” U.S. Pat. No. 6,214,587; and Helen H. Lee et al.,“Nucleic Acid Amplification Technologies: Application To DiseaseDiagnosis” (1997). (Each of the foregoing amplification references ishereby incorporated by reference herein.) Any other amplificationprocedure which meets the definition of “nucleic acid amplification”supra is also contemplated by the inventors.

In one embodiment, amplification oligonucleotides of the presentdisclosure are unlabeled. In another embodiment, amplificationoligonucleotides of the present disclosure include one or more reportergroups to facilitate detection of a target nucleic acid in combinationwith or exclusive of a detection probe. A wide variety of methods areavailable to detect an amplified target sequence. For example, thenucleotide substrates or the amplification oligonucleotides can includea detectable label that is incorporated into newly synthesized DNA. Theresulting labeled amplification product is then generally separated fromthe unused labeled nucleotides or amplification oligonucleotides and thelabel is detected in the separated product fraction. (See, e.g., Wu,“Detection of Amplified Nucleic Acid Using Secondary CaptureOligonucleotides and Test Kit,” U.S. Pat. No. 5,387,510.)

A separation step is not required, however, if the amplificationoligonucleotide is modified by, for example, linking it to aninteracting label pair, such as two dyes which form a donor/acceptor dyepair. The modified amplification oligonucleotide can be designed so thatthe fluorescence of one dye pair member remains quenched by the otherdye pair member, so long as the amplification oligonucleotide does nothybridize to target nucleic acid, thereby physically separating the twodyes. Moreover, the amplification oligonucleotide can be furthermodified to include a restriction endonuclease recognition sitepositioned between the two dyes so that when a hybrid is formed betweenthe modified amplification oligonucleotide and target nucleic acid, therestriction endonuclease recognition site is rendered double-strandedand available for cleavage or nicking by an appropriate restrictionendonuclease. Cleavage or nicking of the hybrid then separates the twodyes, resulting in a change in fluorescence due to decreased quenchingwhich can be detected as an indication of the presence of the targetorganism in the test sample. This type of modified amplificationoligonucleotide, referred to as a “signal primer,” is disclosed byNadeau et al., “Detection of Nucleic Acids by Fluorescence Quenching,”U.S. Pat. No. 6,054,279.

Substances which can serve as useful detectable labels are well known inthe art and include radioactive isotopes, fluorescent molecules,chemiluminescent molecules, chromophores, as well as ligands such asbiotin and haptens which, while not directly detectable, can be readilydetected by a reaction with labeled forms of their specific bindingpartners, e.g., avidin and antibodies, respectively.

Another approach is to detect the amplification product by hybridizationwith a detectably labeled oligonucleotide probe and measuring theresulting hybrids in any conventional manner. In particular, the productcan be assayed by hybridizing a chemiluminescent acridiniumester-labeled oligonucleotide probe to the target sequence, selectivelyhydrolyzing the acridinium ester present on unhybridized probe, andmeasuring the chemiluminescence produced from the remaining acridiniumester in a luminometer. (See, e.g., Arnold et al., U.S. Pat. No.5,283,174, and Norman C. Nelson et al., “Nonisotopic Probing, Blotting,and Sequencing,” ch. 17 (Larry J. Kricka ed., 2d ed. 1995).)

Because genitourinary specimens tend to contain large amounts of T.vaginalis when an individual is infected with the organism, it may bedesirable to include a co-amplifiable pseudo target in the amplificationreaction mixture in order to render the assay less sensitive, especiallywhen quantification is an objective of the assay. Pseudo targets andtheir uses are disclosed by Nunomura, “Polynucleotide AmplificationMethod,” U.S. Pat. No. 6,294,338, the contents of which are herebyincluded by reference herein. In the present application, the pseudotarget may be, for example, a known amount of a Trichomonas tenax 18SrRNA transcript that can be amplified with a set of amplificationoligonucleotides of the present disclosure under amplificationconditions, but which does not contain or result in a sequence that isdetectable with a detection probe of the present disclosure.Alternatively a pseudo target may be a synthetic oligonucleotide thatcan be amplified with a set of amplification oligonucleotides of thepresent disclosure under amplification conditions, but which does notcontain or result in a sequence that is detectable with a detectionprobe of the present disclosure.

D. SAMPLE PROCESSING

Sample processing prior to amplification or detection of a targetsequence may be necessary or useful for discriminating a target sequencefrom non-target nucleic acid present in a sample. Sample processingprocedures may include, for example, direct or indirect immobilizationof nucleic acids and/or oligonucleotides from the liquid phase in aheterogeneous assay. With some procedures, such immobilization mayrequire multiple hybridization events. Ranki et al., “Detection ofMicrobial Nucleic Acids by a One-Step Sandwich Hybridization Test,” U.S.Pat. Nos. 4,486,539 and 4,563,419, for example, disclose a one-stepnucleic acid “sandwich” hybridization method involving the use of asolid-phase bound nucleic acid having a target complementary sequenceand a labeled nucleic acid probe which is complementary to a distinctregion of the target nucleic acid. Stabinsky, “Methods and Kits forPerforming Nucleic Acid Hybridization Assays,” U.S. Pat. No. 4,751,177,discloses methods including a “mediator” polynucleotide that reportedlyovercomes sensitivity problems associated with Ranki s method resultingfrom leakage of immobilized probe from the solid support. Instead ofdirectly immobilizing the target nucleic acid, the mediatorpolynucleotides of Stabinsky are used to bind and indirectly immobilizetarget polynucleotide:probe polynucleotide complexes which have formedfree in solution.

Any known solid support may be used for sample processing, such asmatrices and particles free in solution. The solid support may be, forexample, nitrocellulose, nylon, glass, polyacrylate, mixed polymers,polystyrene, silane polypropylene, or particles having a magnetic chargeto facilitate recovering sample and/or removing unbound nucleic acids orother sample components. In one embodiment the supports are magneticspheres that are monodisperse (i.e., uniform in size±5%), therebyproviding consistent results, which is particularly advantageous for usein an automated procedure. One such automated procedure is disclosed byAmmann et al., “Automated Process for Isolating and Amplifying a TargetNucleic Acid Sequence,” U.S. Pat. No. 6,335,166, the contents of whichare incorporated by reference.

An oligonucleotide for immobilizing a target nucleic acid on a solidsupport may be joined directly or indirectly to the solid support by anylinkage or interaction which is stable under assay conditions (e.g.,conditions for amplification and/or detection). Referred to herein as an“immobilized probe,” this oligonucleotide may bind directly to thetarget nucleic acid or it may include a base sequence region, such as ahomopolymeric tract (e.g., a poly dT) or a simple short repeatingsequence (e.g., an AT repeat), which hybridizes to a complementary basesequence region present on a capture probe. Direct joining occurs whenthe immobilized probe is joined to the solid support in the absence ofan intermediate group. For example, direct joining may be via a covalentlinkage, chelation or ionic interaction. Indirect joining occurs whenthe immobilized probe is joined to the solid support by one or morelinkers. A “linker” is a means for binding at least two differentmolecules into a stable complex and contains one or more components of abinding partner set.

Members of a binding partner set are able to recognize and bind to eachother. Binding partner sets may be, for example, receptor and ligand,enzyme and substrate, enzyme and cofactor, enzyme and coenzyme, antibodyand antigen, sugar and lectin, biotin and streptavidin, ligand andchelating agent, nickel and histidine, substantially complementaryoligonucleotides, and complementary homopolymeric nucleic acids orhomopolymeric portions of polymeric nucleic acids. Components of abinding partner set are the regions of the members that participate inbinding.

A sample processing system having practical advantages in terms of itsease of use and rapidity comprises an immobilized probe containing abase sequence which is complementary to a base sequence of a captureprobe, referred to herein as an “immobilized probe binding region.” Thecapture probe additionally contains a base sequence, referred to hereinas a “target-complementary base sequence,” which may specificallyhybridize to a target sequence contained in a target nucleic acid underassay conditions. (While specificity of the target-complementary basesequence of the capture probe for a region of the target nucleic acid isdesirable to minimize the number of non-target nucleic acids remainingfrom the sample after a separation step, it is not a requirement of thecapture probes of the present disclosure if the capture probes are beingused solely to isolate target nucleic acid.) If the capture probe is notbeing employed to isolate a target nucleic acid for subsequentamplification of a target sequence, the capture probe may furtherinclude a detectable label attached within or near thetarget-complementary base sequence, such as a substituted orunsubstituted acridinium ester. The labeled capture probe may be used ina homogeneous or semi-homogenous assay to specifically detect hybridnucleic acids without detecting single-stranded nucleic acids, such asthe capture probe. A homogenous assay which could be used with thissystem is the hybridization protection assay (HPA), which is discussedabove in the section entitled “Hybridization Conditions and ProbeDesign.” Following the HPA format, label associated with capture probeswhich have not hybridized to target nucleic acids would be hydrolyzedwith the addition of a mild base, while label associated with captureprobe:target hybrids would be protected from hydrolysis.

An advantage of this latter assay system is that only a singletarget-specific hybridization event (capture probe:target) is necessaryfor target detection, rather than multiple such events (e.g., captureprobe:target and probe:target or probe:amplicon) which are required inother sample processing procedures described herein. Also, feweroligonucleotides in an assay tend to make the assay faster and simplerto optimize, since the overall rate at which a target nucleic acid iscaptured and detected is limited by the slowest hybridizingoligonucleotide. While the target-complementary base sequence of acapture probe may be less specific in alternative assay systems, it muststill be rare enough to avoid significant saturation of the captureprobe with non-target nucleic acids. Thus, the requirement that twoseparate and specific target sequences be identified in thesealternative systems could place constraints on the identification of anappropriate target. By contrast, only one such target sequence is neededwhen the capture probe simultaneously functions as the detection probe.

Whichever approach is adopted, the assay needs to include means fordetecting the presence of the target nucleic acid in the test sample. Avariety of means for detecting target nucleic acids are well known tothose skilled in the art of nucleic acid detection, including meanswhich do not require the presence of a detectable label. Other meansincludes using a detectable label. A labeled probe for detecting thepresence of a target nucleic acid would have to include a base sequencewhich is substantially complementary and specifically hybridizes to atarget sequence contained in the target nucleic acid. Once the probestably binds to the target nucleic acid, and the resulting target:probehybrid has been directly or indirectly immobilized, unbound probe can bewashed away or inactivated and the remaining bound probe can be detectedand/or measured.

Sample processing systems combine the elements of detection and nucleicacid amplification. These systems first directly or indirectlyimmobilize a target nucleic acid using a capture probe, the capturedtarget nucleic acid is purified by removing inter alia cellular debris,non-target nucleic acid and amplification inhibitors from thesample-containing vessel, which is followed by amplification of a targetsequence contained in the target nucleic acid. The amplified product, inone embodiment, is then detected in solution with a labeled probe. (Thetarget nucleic acid may remain in the immobilized state duringamplification or it may be eluted from the solid support prior toamplification using appropriate conditions, such as by first incubatingat a temperature above the T_(m) of the capture probe:target complexand/or the T_(m) of the capture probe:immobilized probe complex.) Oneembodiment of this system is disclosed by Weisburg et al., “Two-StepHybridization and Capture of a Polynucleotide,” U.S. Pat. No. 6,110,678.In this system, the capture probe hybridizes to the target nucleic acidand an immobilized probe hybridizes to the capture probe:target complexunder different hybridization conditions. Under a first set ofhybridization conditions, hybridization of the capture probe to thetarget nucleic acid is favored over hybridization of the capture probeto the immobilized probe. Thus, under this first set of conditions, thecapture probe is in solution rather than bound to a solid support,thereby maximizing the concentration of the free capture probe andutilizing favorable liquid phase kinetics for hybridization to thetarget nucleic acid. After the capture probe has had sufficient time tohybridize to the target nucleic acid, a second set of hybridizationconditions is imposed permitting in the capture probe:target complex tohybridize to the immobilized probe, thereby isolating the target nucleicacid in the sample solution. The immobilized target nucleic acid maythen be purified, and a target sequence present in the target nucleicacid may be amplified and detected. A purification procedure whichincludes one or more wash steps is generally desirable when working withcrude samples (e.g., clinical samples) to prevent enzyme inhibitionand/or nucleic acid degradation due to substances present in the sample.

One embodiment of an amplification method is the transcription-mediatedamplification method disclosed by Kacian et al., “Nucleic Acid SequenceAmplification Methods,” U.S. Pat. No. 5,480,789. In accord with thismethod, a promoter-primer having a 3′ region complementary to a portionof the target and a 5′ promoter region and a primer having the samenucleotide sequence as a portion of the target are contacted with atarget RNA molecule. The primer and promoter-primer define theboundaries of the target region to be amplified, including both thesense present on the target molecule and its complement, and thus thelength and sequence of the amplicon. In this embodiment, theamplification oligonucleotides and immobilized target RNA are contactedin the presence of effective amounts of Moloney murine leukemiavirus-derived reverse transcriptase and T7 RNA polymerase, bothribonucleotide and deoxyribonucleotide triphosphates, and necessarysalts and cofactors at 42° C. Under these conditions, nucleic acidamplification occurs, resulting predominantly in the production of RNAamplicons of a sense opposite to that of the target nucleic acid. Theseamplicons can then be detected in solution by, for example, using anacridinium ester-labeled hybridization assay probe of the same sense asthe target nucleic acid, employing HPA, as disclosed by Arnold et al. inU.S. Pat. No. 5,283,174.

The 3′ terminus of the immobilized probe and the capture probe are, inone embodiment, “capped” or blocked to prevent or inhibit their use astemplates for nucleic acid polymerase activity. Capping may involveadding 3′ deoxyribonucleotides (such as cordycepin),3′,2′-dideoxynucleotide residues, non-nucleotide linkers, such as thosedisclosed by Arnold et al. in U.S. Pat. No. 6,031,091, alkane-diolmodifications, or non-complementary nucleotide residues at the 3′terminus.

Those skilled in the art will recognize that the above-describedmethodology is amenable, either as described or with obviousmodifications, to various other amplification schemes, including, forexample, the polymerase chain reaction (PCR), Qβ replicase-mediatedamplification, self-sustained sequence replication (3SR), stranddisplacement amplification (SDA), nucleic acid sequence-basedamplification (NASBA), loop-mediated isothermal amplification (LAMP),and the ligase chain reaction (LCR).

E. CAPTURE PROBES FOR ISOLATING TRICHOMONAS VAGINALIS RIBOSOMAL NUCLEICACID

Capture probes of the present disclosure are designed to bind to andisolate nucleic acid derived from the 18S ribosomal nucleic acid of T.vaginalis in the presence of non-target nucleic acid. As such, thecapture probes, in one embodiment, include both a target-complementarybase sequence and an immobilized probe binding region. Thetarget-complementary base sequence of the capture probes includes a basesequence which hybridizes to a target sequence derived from 18Sribosomal nucleic acid from T. vaginalis under assay conditions. Whilenot essential, the target-complementary base sequence, in oneembodiment, exhibits specificity for the target sequence in the presenceof non-target nucleic acid under assay conditions. The immobilized probebinding region has a base sequence which hybridizes to an immobilizedprobe comprising a polynucleotide, or a chimeric containingpolynucleotide sequences, which is joined to a solid support present inthe test sample, either directly or indirectly. The target-complementarybase sequence and the immobilized probe binding region may be joined toeach other directly or by means of, for example, a nucleotide basesequence, an abasic sequence or a non-nucleotide linker.

In another embodiment of the present disclosure, a capture probe isprovided for extracting target nucleic acid derived from T. vaginalispresent in a test sample. The base sequence of the capture probeconsists of a target-complementary base sequence that is perfectlycomplementary to a target sequence contained within a target domainselected from the group consisting of SEQ ID Nos. 77, 78, 79, 80, or andRNA/DNA combination equivalent to any of the foregoing. Thetarget-complementary base sequence of the capture probe includes thebase sequence of SEQ ID Nos. 81, 82, 83, 81, or an RNA/DNA combinationequivalent to any of the foregoing. The capture probe may also includeat least one base sequence that is non-complementary to the T. vaginalisnucleic acid. In another embodiment, the capture probes contain atarget-complementary base sequence having a base sequence selected fromthe group consisting of SEQ ID Nos. 55, 56, 57, 58, 85, 86, 87, 88, 90,91, 92, 93, 95, 96, 97, 98, 100, 101, 102, 103, or an RNA/DNAcombination equivalent to any of the foregoing. The immobilized probebinding region of these capture probes comprises a base sequence whichhybridizes to an immobilized probe joined directly or indirectly to asolid support provided to the test sample under assay conditions. In oneexample, the immobilized probe binding region comprises a homopolymericregion (e.g., poly dA) located at the 3′ end of the capture probe whichis complementary to a homopolymeric region (e.g., poly dT) located atthe 5′ end of the immobilized probe. The immobilized probe bindingregion may consists of the base sequence of SEQ ID NO:60tttaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa. Other base sequences may beincorporated into the immobilized probe binding region, including, forexample, short repeating sequences.

To prevent undesirable cross-hybridization reactions, the capture probesof the present disclosure, in one embodiment, exclude nucleotide basesequences, other than the nucleotide base sequence of thetarget-complementary base sequence, which can stably bind to nucleicacid derived from any organism which may be present in the test sampleunder assay conditions. Consistent with this approach, and in order tomaximize the immobilization of capture probe:target complexes which areformed, the nucleotide base sequence of the immobilized probe bindingregion is, in one embodiment, designed so that it can stably bind to anucleotide base sequence present in the immobilized probe under assayconditions and not to nucleic acid derived from any organism which maybe present in the test sample.

The target-complementary base sequence and the immobilized probe bindingregion of the capture probe may be selected so that the captureprobe:target complex has a higher T_(m) than the T_(m) of the captureprobe:immobilized probe complex. In this way, a first set of conditionsmay be imposed which favors hybridization of the capture probe to thetarget sequence over the immobilized probe, thereby providing foroptimal liquid phase hybridization kinetics for hybridization of thecapture probe to the target sequence. Once sufficient time has passedfor the capture probe to bind to the target sequence, a second set ofless stringent conditions may be imposed which allows for hybridizationof the capture probe to the immobilized probe.

Capture probes of the present disclosure may also include a label or apair of interacting labels for direct detection of the target sequencein a test sample. Non-limiting examples of labels, combinations oflabels and means for labeling probes are set forth supra in the sectionentitled “Preparation of Oligonucleotides” and infra in the sectionentitled “Detection Probes to Trichomonas vaginalis Ribosomal NucleicAcid.” A particularly useful method for detecting the presence of acapture probe hybridized to a target nucleic acid is the HybridizationProtection Assay (HPA), which is described above in the section entitled“Hybridization Conditions and Probe Design.” HPA is a homogenous assaywhich distinguishes between probe hybridized to target nucleic acid andprobe which remains unhybridized. Signal detected from an HPA reactionvessel provides an indication of the presence or amount of targetorganisms in the test sample.

Despite their application in a direct detection assay, the most commonuse of capture probes is in the isolation and purification of targetnucleic acid prior to amplifying a target sequence contained in thetarget nucleic acid. By isolating and purifying the target nucleic acidprior to amplification, the number of unintended amplification reactions(i.e., amplification of non-target nucleic acid) can be severelylimited. And, to prevent or inhibit the capture probe itself fromfunctioning as a template for nucleic acid polymerase activity in thepresence of amplification reagents and under amplification conditions,the 3′ end of the capture probe may be capped or blocked. Examples ofcapping agents include 3′ deoxyribonucleotides, 3′,2′-dideoxynucleotideresidues, non-nucleotide linkers, alkane-diol modifications, andnon-complementary nucleotide residues at the 3′ terminus.

F. DETECTION PROBES TO TRICHOMONAS VAGINALIS RIBOSOMAL NUCLEIC ACID

This embodiment of the disclosure relates to novel detection probes.Hybridization is the association of two single strands of complementarynucleic acid to form a hydrogen-bonded double strand. A nucleic acidsequence able to hybridize to a nucleic acid sequence sought to bedetected (“target sequence”) can serve as a probe for the targetsequence. Hybridization may occur between complementary nucleic acidstrands, including DNA/DNA, DNA/RNA, and RNA/RNA, as well as betweensingle-stranded nucleic acids wherein one or both strands of theresulting hybrid contain at least one modified nucleotide, nucleoside,nucleobase, and/or base-to-base linkage. In any case, two single strandsof sufficient complementarity may hybridize to form a double-strandedstructure in which the two strands are held together by hydrogen bondsbetween pairs of complementary bases. As described above, in general Ais hydrogen-bonded to T or U, while G is hydrogen-bonded to C. At anypoint along the hybridized strands, therefore, the classical base pairsAT or AU, TA or UA, GC, or CG may be found. Thus, when a first singlestrand of nucleic acid contains sufficient contiguous complementarybases to a second, and those two strands are brought together underconditions that promote their hybridization, double-stranded nucleicacid will result. Accordingly, under appropriate conditions,double-stranded nucleic acid hybrids may be formed.

The rate and extent of hybridization is influenced by a number offactors. For instance, it is implicit that if one of the two strands iswholly or partially involved in a hybrid, it will be less able toparticipate in the formation of a new hybrid. By designing a probe sothat a substantial portion of the sequence of interest issingle-stranded, the rate and extent of hybridization may be greatlyincreased. Also, if the target is an integrated genomic sequence it willnaturally occur in a double-stranded form, as is the case with a productof PCR. These double-stranded targets are naturally inhibitory tohybridization with a single-stranded probe and require denaturation (inat least the region to be targeted by the probe) prior to thehybridization step. In addition, there can be intra-molecular andinter-molecular hybrids formed within a probe if there is sufficientself-complementarity. Regions of the nucleic acid known or expected toform strong internal structures inhibitory to hybridization are lesspreferred. Examples of such structures include hairpin loops. Likewise,probes with extensive self-complementarity generally should be avoided.All these undesirable structures can be avoided through careful probedesign, and commercial computer programs are available to search forthese types of interactions, such as the Oligo Tech analysis software.

In some applications, probes exhibiting at least some degree ofself-complementarity are desirable to facilitate detection ofprobe:target duplexes in a test sample without first requiring theremoval of unhybridized probe prior to detection. Molecular torch probesare a type of self-complementary probes that are disclosed by Becker etal., “Molecular Torches,” U.S. Pat. No. 6,361,945. The molecular torchprobes disclosed Becker et al. have distinct regions ofself-complementarity, referred to as “the target binding domain” and“the target closing domain,” which are connected by a joining region andwhich hybridize to one another under predetermined hybridization assayconditions. When exposed to denaturing conditions, the complementaryregions (which may be fully or partially complementary) of the moleculartorch probe melt, leaving the target binding domain available forhybridization to a target sequence when the predetermined hybridizationassay conditions are restored. And when exposed to strand displacementconditions, a portion of the target sequence binds to the target bindingdomain and displaces the target closing domain from the target bindingdomain. Molecular torch probes are designed so that the target bindingdomain favors hybridization to the target sequence over the targetclosing domain. The target binding domain and the target closing domainof a molecular torch probe include interacting labels (e.g.,luminescent/quencher) positioned so that a different signal is producedwhen the molecular torch probe is self-hybridized as opposed to when themolecular torch probe is hybridized to a target nucleic acid, therebypermitting detection of probe:target duplexes in a test sample in thepresence of unhybridized probe having a viable label or labelsassociated therewith.

Another example of detection probes having self-complementarity are themolecular beacon probes disclosed by Tyagi et al. in U.S. Pat. No.5,925,517. Molecular beacon probes include nucleic acid molecules havinga target complement sequence, an affinity pair (or nucleic acid arms)holding the probe in a closed conformation in the absence of a targetnucleic acid sequence, and a label pair that interacts when the probe isin a closed conformation. Hybridization of the target nucleic acid andthe target complement sequence separates the members of the affinitypair, thereby shifting the probe to an open confirmation. The shift tothe open confirmation is detectable due to reduced interaction of thelabel pair, which may be, for example, a fluorophore and quencher, suchas DABCYL and EDANS.

The rate at which a probe hybridizes to its target is one measure of thethermal stability of the target secondary structure in the probe region.The standard measurement of hybridization rate is the C_(o)t_(1/2),which is measured as moles of nucleotide per liter times seconds. Thus,it is the concentration of probe times the time at which 50% of maximalhybridization occurs at that concentration. This value is determined byhybridizing various amounts of probe to a constant amount of target fora fixed time. The C_(o)t_(1/2) is found graphically by standardprocedures. The probe:target hybrid melting temperature may bedetermined by isotopic methods well-known to those skilled in the art.The melting temperature (T_(m)) for a given hybrid will vary dependingon the hybridization solution being used.

In one embodiment, detection probes are sufficiently complementary tothe target nucleic acid sequence to hybridize therewith under stringenthybridization conditions. Examples of stringent conditions include atemperature of about 60° C. and a salt concentration of about 1.5 M.Examples of salts include, but are not limited to, lithium chloride,sodium chloride and sodium citrate.

Thus, in a first aspect, the present disclosure features detectionprobes able to distinguish T. vaginalis-derived nucleic acid from non-T.vaginalis nucleic acid (e.g., Trichomonas tenax) by virtue of theability of the detection probe to preferentially hybridize to T.vaginalis-derived nucleic acid) under stringent hybridizationconditions. Specifically, the detection probes contain anoligonucleotide having a base sequence that is substantiallycomplementary to a target sequence present in T. vaginalis-derivednucleic acid.

In the case of a hybridization assay, the length of the target nucleicacid sequence and, accordingly, the length of the probe sequence can beimportant. In some cases, there may be several sequences from aparticular region, varying in location and length, which will yieldprobes with the desired hybridization characteristics. In other cases,one sequence may have better hybridization characteristics than anotherthat differs merely by a single base. While it is possible for nucleicacids that are not perfectly complementary to hybridize, the longeststretch of perfectly homologous base sequence will normally primarilydetermine hybrid stability. While probes of different lengths and basecomposition may be used, the probes in the present disclosure, are up to100 bases in length in one embodiment, or are from 25 to 50 bases inlength in another embodiment, or are from 25 to 35 bases in length inyet another embodiment.

The detection probes include a base sequence that is substantiallycomplementary to a target sequence present in 18S ribosomal RNA (rRNA),or the encoding DNA (rDNA), of T. vaginalis. Thus, the detection probesare able to stably hybridize to a target sequence derived from T.vaginalis under stringent hybridization conditions. The detection probesmay also have additional bases outside of the targeted nucleic acidregion which may or may not be complementary to T. vaginalis-derivednucleic acid but which are not complementary to nucleic acid derivedfrom a non-target organism which may be present in the test sample.

Probes (and amplification oligonucleotides) of the present disclosuremay also be designed to include a capture tail comprised of a basesequence (distinct from the base sequence intended to hybridize to thetarget sequence) that can hybridize under predetermined hybridizationconditions to a substantially complementary base sequence present in animmobilized oligonucleotide that is joined to a solid support. Theimmobilized oligonucleotide is, in one embodiment, joined to amagnetically charged particle that can be isolated in a reaction vesselduring a purification step after a sufficient period of time has passedfor probe to hybridize to target nucleic acid. (An example of aninstrument which can be used to perform such a purification step is theDTS® 1600 Target Capture System (Gen-Probe; Cat. No. 5202).) The probeis, in one embodiment, designed so that the melting temperature of theprobe:target hybrid is greater than the melting temperature of theprobe:immobilized oligonucleotide hybrid. In this way, different sets ofhybridization assay conditions can be employed to facilitatehybridization of the probe to the target nucleic acid prior tohybridization of the probe to the immobilized oligonucleotide, therebymaximizing the concentration of free probe and providing favorableliquid phase hybridization kinetics. This “two-step” target capturemethod is disclosed by Weisburg et al., “Two Step Hybridization andCapture of a Polynucleotide,” U.S. Pat. No. 6,110,678, the contents ofwhich are hereby incorporated by reference herein. Other target captureschemes which could be readily adapted to the present disclosure arewell known in the art and include, for example, those disclosed by Rankiet al., “Detection of Microbial Nucleic Acids by a One-Step SandwichHybridization Test,” U.S. Pat. No. 4,486,539, and Stabinsky, “Methodsand Kits for Performing Nucleic Acid Hybridization Assays,” U.S. Pat.No. 4,751,177.

For T. vaginalis detection probes, the terms “target nucleic acidsequence,” “target nucleotide sequence,” “target sequence,” and “targetregion” all refer to a nucleic acid sequence present in T. vaginalisrRNA or rDNA, or a sequence complementary thereto, which is notidentically present in the nucleic acid of a closely related species.Nucleic acids having nucleotide sequences complementary to a targetsequence may be generated by target amplification techniques disclosedelsewhere herein.

Organisms closely related to T. vaginalis include Trichomonas gallinae,Trichomonas tenax, Monotrichomonas species ATCC No. 50693, Ditrichomonashonigbergi, Tritrichomonas foetus, Tetratrichomonas gallinarum andPentatrichomonas hominis, with Trichomonas tenax being the most closelyrelated. In addition to these organisms, organisms that might beexpected to be present in a T. vaginalis-containing test sample include,for example, Escherichia coli, Chlamydia trachomatis and Neiserriagonorrhoeae. This list of organisms is by no means intended to be fullyrepresentative of the organisms that the T. vaginalis detection probesof the present disclosure can be used to distinguish over. In general,the T. vaginalis detection probes of the present disclosure can be usedto distinguish T. vaginalis-derived nucleic acid from any non-T.vaginalis nucleic acid that does not stably hybridize with the probe(s)under stringent hybridization conditions. Examples of detection probesare listed in Table 2.

TABLE 2  T. vaginalis 18S Detection Probes SEQ ID NO. Sequence 1ttgccgaagtccttcggttaaagttctaattg 2 uugccgaaguccuucgguuaaaguucuaauug 3caattagaactttaaccgaaggacttcggcaa 4 caauuagaacuuuaaccgaaggacuucggcaa 5tgccgaagtccttcggttaaagttctaattgg 6 ugccgaaguccuucgguuaaaguucuaauugg 7ccaattagaactttaaccgaaggacttcggca 8 ccaauuagaacuuuaaccgaaggacuucggca 9gccgaagtccttcggttaaagttctaattggg 10 gccgaaguccuucgguuaaaguucuaauuggg 11cccaattagaactttaaccgaaggacttcggc 12 cccaauuagaacuuuaaccgaaggacuucggc 13ccgaagtccttcggttaaagttctaattggg 14 ccgaaguccuucgguuaaaguucuaauuggg 15cccaattagaactttaaccgaaggacttcgg 16 cccaauuagaacuuuaaccgaaggacuucgg 17cgaagtccttcggttaaagttctaattgggac 18 cgaaguccuucgguuaaaguucuaauugggac 19gtcccaattagaactttaaccgaaggacttcg 20 gucccaauuagaacuuuaaccgaaggacuucg 21cgaagtcittcggttaaagttctaattgggac 22 cgaaguciuucgguuaaaguucuaauugggac 23gtcccaattagaactttaaccgaaigacttcg 24 gucccaauuagaacuuuaaccgaaigacuucg 25gaaguccuucgguuaaaguucuaa 26 gaaguccuucgguuaaaguucuaa 27ttagaactttaaccgaaggacttc 28 uuagaacuuuaaccgaaggacuuc 29gtccttcggttaaagttctaattgg 30 guccuucgguuaaaguucuaauugg 31ccaattagaactttaaccgaaggac 32 ccaauuagaacuuuaaccgaaggac 33ttcggttaaagttctaattgggactccctgcg 34 uucgguuaaaguucuaauugggacucccugcg 35cgcagggagtcccaattagaactttaaccgaa 36 cgcagggagucccaauuagaacuuuaaccgaa

In one embodiment, detection probes were designed around the 1150 baseregion of T. vaginalis 18S ribosomal RNA, GenBank accession numberU17510.1 and GI number 687613. The T. vaginalis detection probes have abase sequence comprising, overlapping with, consisting essentially of,consisting of, substantially corresponding to, or contained within abase sequence of SEQ ID Nos. 1-36. The detection probes may include anacridinium ester label joined to the probes by means of a non-nucleotidelinker positioned between nucleotides 6 and 7, 7 and 8, 10 and 11, 11and 12, 13 and 14, 14 and 15, 15 and 16, 16 and 17, 17 and 18, 18 and19, or 19 and 20. The acridinium ester label may be joined to the probein accordance with the teachings of Arnold et al. in U.S. Pat. Nos.5,185,439 and 6,031,091.

Once synthesized, the probes may be labeled with a detectable label orreporter group by any well-known method. (See, e.g., Sambrook et al.,supra, ch. 10.) The probe may be labeled with a detectable moiety suchas a radioisotope, antigen or chemiluminescent moiety to facilitatedetection of the target sequence. Useful labels include radioisotopes aswell as non-radioactive reporting groups. Isotopic labels include ³H,³⁵S, ³²P, ¹²⁵I, ⁵⁷Co and ¹⁴C. Isotopic labels can be introduced into anoligonucleotide by techniques known in the art such as nick translation,end labeling, second strand synthesis, reverse transcription and bychemical methods. When using radiolabeled probes, hybridization can bedetected by techniques such as autoradiography, scintillation countingor gamma counting. The chosen detection method depends on the particularradioisotope used for labeling.

Non-isotopic materials can also be used for labeling and may beintroduced internally between nucleotides or at an end of theoligonucleotide. Modified nucleotides may be incorporated enzymaticallyor chemically. Chemical modifications of the oligonucleotide may beperformed during or after synthesis of the oligonucleotide usingtechniques known in the art. For example, through use of non-nucleotidelinker groups disclosed by Arnold et al. in U.S. Pat. No. 6,031,091.Non-isotopic labels include fluorescent molecules, chemiluminescentmolecules, fluorescent chemiluminescent molecules, phosphorescentmolecules, electrochemiluminescent molecules, chromophores, enzymes,enzyme cofactors, enzyme substrates, dyes and haptens or other ligands.Another useful labeling technique is a base sequence that is unable tostably hybridize to the target nucleic acid under stringent conditions.Probes of the present disclosure are, in one embodiment, labeled with anacridinium ester. (Acridinium ester labeling is disclosed by Arnold etal. in U.S. Pat. No. 5,185,439.)

The selected detection probe can then be brought into contact with atest sample suspected of containing T. vaginalis. Generally, the testsample is from a source that also contains unknown organisms. Typically,the source of the test sample will be a patient specimen, such as agenitourinary specimen. After bringing the probe into contact withnucleic acids derived from the test sample, the probe and sample-derivednucleic acids can be incubated under conditions permitting preferentialhybridization of the probe to a target nucleic acid derived from T.vaginalis that may be present in the test sample in the presence ofnucleic acid derived from other organisms present in the test sample.

After a detection probe has hybridized to target nucleic acid present inthe test sample, the resulting hybrid may be separated and detected byvarious techniques well known in the art, such as hydroxyapatiteadsorption and radioactive monitoring. Other techniques include thosewhich involve selectively degrading label associated with unhybridizedprobe and then measuring the amount of remaining label associated withhybridized probe, as disclosed in U.S. Pat. No. 5,283,174. The inventorsparticularly prefer this latter technique.

EXAMPLES

Examples are provided below illustrating different aspects andembodiments of the disclosure. It is believed that these examplesaccurately reflect the details of experiments actually performed,however, it is possible that some minor discrepancies may exist betweenthe work actually performed and the experimental details set forth belowwhich do not affect the conclusion of these experiments. Skilledartisans will appreciate that these examples are not intended to limitthe disclosure to the specific embodiments described therein.

Reagents

Various reagents are identified in the examples below, the formulationsand pH values (where relevant) of these reagents were as follows.

A “Lysis Buffer” contains 15 mM sodium phosphate monobasic monohydrate,15 mM sodium phosphate dibasic anhydrous, 1.0 mM EDTA disodiumdihydrate, 1.0 mM EGTA free acid, and 110 mM lithium lauryl sulfate, pH6.7.

A “Urine Lysis Buffer” contains 150 mM HEPES free acid, 294 mM lithiumlauryl sulfate, 57 mM lithium hydroxide monohydrate, 100 mM ammoniumsulfate, pH 7.5.

A “Target Capture Reagent” contains 250 mM HEPES free acid dihydrate,310 mM lithium hydroxide monohydrate, 1.88 M lithium chloride, 100 mMEDTA free acid, 2 M lithium hydroxide to pH 6.4, and 250 μg/ml 1 micronmagnetic particles Sera-Mag™ MG-CM Carboxylate Modified (Seradyn, Inc.;Indianapolis, Ind.; Cat. No. 24152105-050450) having oligo(dT)₁₄covalently bound thereto.

A “Wash Solution” contains 10 mM HEPES free acid, 6.5 mM sodiumhydroxide, 1 mM EDTA free acid, 0.3% (v/v) ethyl alcohol absolute, 0.02%(w/v) methyl paraben, 0.01% (w/v) propyl paraben, 150 mM sodiumchloride, 0.1% (w/v) lauryl sulfate, sodium (SDS), and 4 M sodiumhydroxide to pH 7.5.

An “Amplification Reagent” is a lyophilized form of a 3.6 mL solutioncontaining 26.7 mM rATP, 5.0 mM rCTP, 33.3 mM rGTP and 5.0 mM rUTP, 125mM HEPES free acid, 8% (w/v) trehalose dihydrate, 1.33 mM dATP, 1.33 mMdCTP, 1.33 mM dGTP, 1.33 mM dTTP, and 4 M sodium hydroxide to pH 7.5.The Amplification Reagent is reconstituted in 9.7 mL of “AmplificationReagent Reconstitution Solution” described below.

An “Amplification Reagent Reconstitution Solution” contains 0.4% (v/v)ethyl alcohol absolute, 0.10% (w/v) methyl paraben, 0.02% (w/v) propylparaben, 33 mM KCl, 30.6 mM MgCl₂, 0.003% phenol red.

A “Primer Reagent” contains 1 mM EDTA disodium dihydrate, ACS, 10 mMTrizma base, and 6M hydrochloric acid to pH 7.5.

An “Enzyme Reagent” is a lyophilized form of a 1.45 mL solutioncontaining 20 mM HEPES free acid dihydrate, 125 mM N-acetyl-L-cysteine,0.1 mM EDTA disodium dihydrate, 0.2% (v/v) TRITON® X-100 detergent, 0.2M trehalose dihydrate, 0.90 RTU/mL Moloney murine leukemia virus(“MMLV”) reverse transcriptase, 0.20 U/mL T7 RNA polymerase, and 4Msodium hydroxide to pH 7.0. (One “unit” or “RTU” of activity is definedas the synthesis and release of 5.75 fmol cDNA in 15 minutes at 37° C.for MMLV reverse transcriptase, and for T7 RNA polymerase, one “unit” or“U” of activity is defined as the production of 5.0 fmol RNA transcriptin 20 minutes at 37° C.) The Enzyme Reagent is reconstituted in 3.6 mLof “Enzyme Reagent Reconstitution Solution” described below.

An “Enzyme Reagent Reconstitution Solution” contains 50 mM HEPES freeacid, 1 mM EDTA free acid, 10% (v/v) TRITON X-100 detergent, 120 mMpotassium chloride, 20% (v/v) glycerol anhydrous, and 4 M sodiumhydroxide to pH 7.0.

A “Probe Reagent” is a lyophilized form of a 3.6 mL solution containing110 mM lithium lauryl sulfate, 10 mM of mercaptoethane sulfonic acid,100 mM lithium succinate, and 3% PVP. The Probe Reagent is reconstitutedin 36 mL of “Probe Reagent Reconstitution Solution” described below.

A “Probe Reagent Reconstitution Solution” contains 100 mM succinic acid,73 mM lithium lauryl sulfate, 100 mM lithium hydroxide monohydrate, 15mM aldrithiol, 1.2 M lithium chloride, 20 mM EDTA, 3% (v/v) ethylalcohol, and 2M lithium hydroxide to pH 4.7.

A “Selection Reagent” contains 600 mM boric acid, ACS, 182.5 mM sodiumhydroxide, ACS, 1% (v/v) TRITON X-100 detergent, and 4 M sodiumhydroxide to pH 8.5.

A “Detection Reagents” comprises Detect Reagent I, which contains 1 mMnitric acid and 32 mM hydrogen peroxide, 30% (v/v), and Detect ReagentII, which contains 1.5 M sodium hydroxide.

An “Oil Reagent” is a silicone oil.

Oligonucleotide Synthesis

The oligonucleotides used in the following examples were synthesizedusing standard phosphoramidite chemistry, in accordance with theteachings of Caruthers et al, Methods Enzymol., 154:287 (1987). Thedetection probe sequences were labeled with a 2-methoxyl acridiniumester, 9[[4-[3-[(2,5-dioxo-1-pyrrolidinyl)oxy]-3-oxopropyl]phenoxy]carbonyl]-2,10-dimethyl-acridiniumtrifluoromethane sulfonate, using the labeling method disclosed inArnold, et al., U.S. Pat. No. 5,185,439. The acridinium ester (AE) wasincorporated into the detection probe sequence via a non-nucleotidelinker in accordance with the teachings of Arnold, et al., U.S. Pat.Nos. 5,585,481, 5,639,604, and 6,031,091, the contents of which arehereby incorporated by reference herein.

In Vitro Transcript

Unless otherwise indicated, the oligonucleotidess in the followingExamples were evaluated using purified in vitro transcript (IVT). TheIVT was made by cloning a portion of the T. vaginalis 18S ribosomal RNAinto a vector and then using the vector to transform cells. Briefly,total RNA from T. vaginalis strains ATCC No. 30488 and ATCC No. 30001was amplified by reverse transcription polymerase chain reaction(RT-PCR) using amplification oligonucleotides having the base sequencesof SEQ ID Nos. 69, 70, 73, and 74 which target a region of the 18S rRNA.The RT-PCR amplicons produced were initially cloned using pGEM®-T EasyVector System II (Promega; Madison, Wis.; Cat. No. A1380). The clonedsequences were excised from the pGEM-T Easy vectors and recloned intopBluescript® II SK (+) vectors (Stratagene; La Jolla, Calif.; Cat. No.212205) using restriction enzymes ApaI and Sac I. The pBluescriptvectors were used to transform XL1 Blue Supercompentant Cells(Stratagene; Cat. No. 200236).

Example 1: Signal-to-Noise Ratios for Detection Probes Targeting the1150 Region of T. vaginalis 18S rRNA

In this example the light off kinetics for several AE-labeled detectionprobes were evaluated. Oligonucleotides having the base sequences of SEQID Nos. 75 and 76 were synthesized using 2′-O-Methyl RNA bases, allother oligonucleotides were synthesized using DNA. For each AE-labeleddetection probe, the target sequence and linker position are indicatedin Table 3 below. The “linker position” identifies the bases betweenwhich the linker is incorporated in the probe sequence.

A probe having the nucleotide sequence of SEQ ID NO:9 was disclosed inWeisburg, et al., U.S. Pat. No. 7,381,811.

TABLE 3  1150 Region Probe Sequences and Linker Positions SEQ Linker IDPosi- Probe NO. Probe Sequence tion A 1 ttgccgaagtccttcggttaaagttctaattg17/18 B 1 ttgccgaagtccttcggttaaagttctaattg 18/19 C 1ttgccgaagtccttcggttaaagttctaattg 19/20 D 5tgccgaagtccttcggttaaagttctaattgg 16/17 E 5tgccgaagtccttcggttaaagttctaattgg 17/18 F 5tgccgaagtccttcggttaaagttctaattgg 18/19 G 9gccgaagtccttcggttaaagttctaattggg 15/16 H 9gccgaagtccttcggttaaagttctaattggg 16/17 I 9gccgaagtccttcggttaaagttctaattggg 17/18 J 13ccgaagtccttcggttaaagttctaattggg 14/15 K 17cgaagtccttcggttaaagttctaattgggac 13/14 L 17cgaagtccttcggttaaagttctaattgggac 14/15 M 17cgaagtccttcggttaaagttctaattgggac 15/16 N 75 gaaguccuucgguuaaaguucuaa 8/9O 75 gaaguccuucgguuaaaguucuaa 13/14 P 75 gaaguccuucgguuaaaguucuaa 14/15Q 76 guccuucgguuaaaguucuaauugg 10/11 R 76 guccuucgguuaaaguucuaauugg11/12 S 76 guccuucgguuaaaguucuaauugg 16/17 T 33ttcggttaaagttctaattgggactccctgcg 6/7 U 33ttcggttaaagttctaattgggactccctgcg 7/8

The detection probes were tested to determine their signal-to-noiseratio using the Hybridization Protection Assay (HPA), in accordance withthe teachings of Arnold et al., U.S. Pat. No. 5,283,174. Briefly, eachlabeled probe was diluted to 1.05e6 relative light units (RLU) per 100microliters (μL) of Probe Reagent and 100 μL of the diluted labeledprobe were added to a 12 millimeter (mm)×75 mm tube. ReconstitutedAmplification Reagent (75 μL) with or without 0.5 picomoles (pmol) ofprobe complement (SEQ ID NO:62 for all probe sequences except SEQ IDNO:33, which used SEQ ID NO:63) was added to the appropriate tubes.Enzyme Reagent (25 μL) was also added to each tube. Oil Reagent (200 μL)was added to prevent evaporation and the tubes were incubated for 20minutes at 62° C. to allow the labeled probes to hybridize to the probecomplement, if present. Label associated with non-hybridized probes wasinactivated by adding 250 μL of Selection Reagent and incubating at 62°C. for 10 minutes. The tubes were cooled to room temperature for 15minutes. The tubes were analyzed in a LEADER luminometer (Gen-ProbeIncorporated; San Diego, Calif.) that was capable of automaticallyinjecting 200 μL of Detect Reagent I followed by 200 μL of DetectReagent II, and then repeatedly reading emission light in the tubes fora specified period of time. For all probes except Probe T and Probe U,five replicates were run with the probe complement (SEQ ID NO:62) andfive replicates were run without the probe complement. For Probe T andProbe U, ten replicates were run with the probe complement (SEQ IDNO:63) and ten replicates were run without the probe complement. Theresults were measured in RLU. The signal-to-noise ratios for thedetection probes were compared to signal-to-noise ratios for the controlsample and control probe, both of which used non-T. vaginalis targetsequences. The detection probes were tested in four different batchesand a control sample and control probe were run with each batch. Thecontrol sample and control probe used in each batch contained the samenon-T. vaginalis target sequence. Probes A, B, C, and I were tested inbatch 1; Probes D, E, F, G, H, I, K, L, and M were tested in batch 2;Probes N, O, P, Q, R, and S were tested in batch 3; and Probes T and Uwere tested in batch 4. The results are summarized in Table 4 below andindicate that Probes B, G, K, L, M, N, and T had signal-to-noise ratiosthat were comparable or better than the control. The coefficient ofvariance (CV) is expressed as a percentage.

TABLE 4 Signal to Noise Ratio Results Detection Negative PositiveSignal-to- Probe Ave. RLU % CV Ave. RLU % CV Noise Ratio Control 6,67948  1,662,158 2 249 A 25,583  4 534,363 2  21 B 2,019 5 636,015 1 315 C3,433 27  615,569 3 179 J 35,634  5 669,623 3  19 Control 2,686 41,115,418 2 415 D 21,934  11  723,867 4  33 E 2,475 26  772345 4 312 F2,370 18  632,216 6 267 G 2,136 16  827,947 3 388 H 3,024 22  704,997 2233 I 4,066 25  882,684 3 217 K 1,688 12  854,857 4 507 L 2,194 14 1,034,094 5 471 M 2,136 16  827,947 3 388 Control 3,550 24  1,069,428 5301 N 3,406 3 1,083,026 8 318 O 1,104 8 9,507 6  9 P 1,997 4 178,286 14 89 Q 4,381 7 53,409 3  12 R 1,856 3 47,700 5  26 S 2,189 3 14,000 4  6Control* 4,163 8 1,577,309 1 378 T*  865 7 936,304 1 1,082  U*  11,134**181** 970,856 3   87**   1,085***  14***   895*** *ten replicates run**2 out of 10 replicates had extra enzyme added due to a mechanicalerror ***Statistics were recalculated without the two outliers

Example 2:Cross-Reactivity of Probes Targeting the 1150 Region of T.vaginalis 18S rRNA with Trichomonas tenax

In this example, the specificity of several detection probes evaluatedin Example 1 were further evaluated to determine their cross-reactivitywith Trichomonas tenax, which is the most closely related protozoa to T.vaginalis and therefore most likely to cross-react with the T. vaginalisprobes. The AE incorporation site for each detection probe was the sameas those listed in Table 3 above. The detection probes were evaluatedusing the same method described in Example 1 with one modification, theprobe complement in the Amplification Reagent was replaced with 0.5 pmolof T. tenax in vitro transcript (IVT). Trichomonas tenax IVT was made byamplifying total RNA from T. tenax cells (ATCC No. 30207) using RT-PCRwith SEQ ID Nos. 69 and 74. The RT-PCR amplicons were cloned intopCR-Script® Amp SK(+) vectors (Stratagene). The vectors were used totransform XL10-Gold® Ultracompetent Cells (Stratagene). For each probe,five replicates were run with T. tenax IVT and five replicates were runwithout T. tenax IVT. The detection probes were compared to the samecontrol probe sequence and control sample that was used in Example 1.The detection probes were tested in two different batches and thecontrol was run for each batch. Probes K and L were tested in batch 1and Probes N, O, P, Q, R, and S tested in batch 2. The results aresummarized in Table 5 below and indicate that the detection probestested in this experiment did not cross-react with the T. tenax IVT.

TABLE 5 Cross-Reactivity With T. tenax Detection Negative PositiveSignal-to- Probe Ave. RLU % CV Ave. RLU % CV Noise Ratio Control 1,17718 963,684 1 819 K 831 5 1,074 13 1 L 771 5 983 2 1 Control 4,960 231,199,459 5 242 N 660 5 7,914 4 12 O 969 17 1,276 8 1 P 610 4 2,063 5 3Q 756 4 20,040 1 27 R 761 4 7,899 6 10 S 852 2 7,427 2 9

Example 3: Detection of In Vitro Transcripts Derived from Two Strains ofT. vaginalis with T. vaginalis Detection Probe

In this example, Probe L (SEQ ID NO:17, AE incorporated using a linkerpositioned between bases 14 and 15) was evaluated to determine itsability to detect two T. vaginalis strains, ATCC Nos. 30488 and 30001.Probe L was combined with a target capture probe (SEQ ID NO:59) andamplification oligonucleotides (SEQ ID Nos. 45, 53, and 61). SEQ ID Nos.59, 45, and 53 are disclosed in Weisburg, et al., U.S. Pat. No.7,381,811. Probe L was evaluated using: (1) Target Capture, described inWeisburg et al., U.S. Pat. No. 6,110,678; (2) Transcription-MediatedAmplification (TMA), described in Kacian et al. in U.S. Pat. Nos.5,399,491 and 5,480,784 (the contents of which are incorporated byreference) and by Lee et al., supra, ch. 8; and (3) HybridizationProtection Assay (HPA), described in Arnold et al., U.S. Pat. No.5,283,174. The protocols for each method are briefly described below.

IVT derived from these strains and stored in Lysis Buffer were dilutedwith Lysis Buffer to 100e6, 75e6, 50e6, 25e6, 10e6 and 0 copies permilliliter (mL) and 400 μL of each concentration were placed in separate12 mm×75 mm tubes. Target Capture Reagent, 100 μL containing 1 nanomole(nmol) per liter (L) of SEQ ID NO:59, was added to each tube and thetubes were covered and incubated at 62° C. for 30 minutes to immobilizethe IVT, if present, on the magnetic beads. The magnetic beads werepelleted using a DTS® 400 Target Capture System (Gen-Probe; Cat. No.104555) and the supernatant was aspirated. The magnetic beads wereresuspended in 1 mL of Wash Solution, re-pelletted and the Wash Solutionwas aspirated. The magnetic beads were resuspended in 75 μL ofreconstituted Amplification Reagent containing 53 pmol/mL of SEQ IDNO:45; 53 pmol/mL of SEQ ID Nos. 53; and 4.4 pmol/L of SEQ ID NO:61. OilReagent (200 μL) was added to prevent evaporation and the tubes werecovered and incubated at 62° C. for 10 minutes to disrupt secondarystructures of the transcripts and allow the primer to bind. The tubeswere then incubated at 42° C. for 5 minutes to bring them to theappropriate temperature for the enzymes. Reconstituted Enzyme Reagent(25 μL) was added and the tubes were incubated at 42° C. for 60 minutesto allow the enzymes to amplify the target nucleic acid. Probe Reagent(100 μL) containing 2e6 RLU of Probe L was added to each tube. The tubeswere briefly vortexed, covered, and incubated at 62° C. for 20 minutesto allow the probe to hybridize to the amplified nucleic acid. The tubeswere incubated at room temperature for 5 minutes. Label on thenon-hybridized probes was inactivated by adding 250 μL of SelectionReagent and incubating at 62° C. for 10 minutes. The tubes were cooledat room temperature for 15 minutes. The tubes were analyzed in a LEADERluminometer capable of automatically injecting 200 μL of Detect ReagentI, followed by 200 μL of Detect Reagent II, and then repeatedly readingthe emission light in the tubes. Five replicates were run for each IVTconcentration. The results were measured in RLU and a minimum of 100,000RLU was the threshold for a test to be considered positive. The resultsare summarized in Table 6 below and indicate that Probe L performed verydifferently between the two T. vaginalis strains. For the ATCC No. 30001strain, Probe L detected every concentration at over 1 million RLU. Forthe ATCC No. 30488 strain, Probe L showed a gradient effect correlatingwith the concentration of IVT. Plus, the RLU at the highestconcentration of ATCC No. 30488 was roughly half of the RLU at thelowest concentration of ATCC No. 3011. This suggests that Probe L is notas sensitive at detecting the ATCC No. 30488 strain as it is atdetecting the ATCC No. 30001 strain.

TABLE 6 Detection of T. vaginalis Strains Using Probe L ATCC No. 30001ATCC No. 30488 IVT Amt. Ave. RLU % CV Ave. RLU % CV 0 3,665 8 1,693 510e6 1,465,270 3 127,006 9 25e6 1,468,481 3 212,967 21 50e6 1,487,129 2464,798 7 75e6 1,393,655 2 490,382 13 100e6  1,198,562 4 714,740 7

Example 4: Detection of In Vitro Transcripts Derived from Two Strains ofT. vaginalis with T. vaginalis Detection Probe

In this example, Probe T (SEQ ID NO:33, AE incorporated using a linkerpositioned between bases 6 and 7) was evaluated to determine its abilityto detect two T. vaginalis strains, ATCC No. 30488 and ATCC No. 30001.Probe T was combined with three amplification oligonucleotides (SEQ IDNos. 45, 53, and 61) and evaluated using TMA and HPA.

IVT derived from these strains and stored in Lysis Buffer were dilutedwith reconstituted Amplification Reagent to 140e6, 10e6, 1e6 and 0copies per 187.5 μL. The diluted IVT (75 μL) was added to individual 12mm×75 mm tubes. Amplification oligonucleotides were added to each tubefor a final concentration of 53 pmol/mL of SEQ ID NO:45; 53 pmol/mL ofSEQ ID Nos. 53; and 4.4 pmol/L of SEQ ID NO:61 in 75 μL. Oil Reagent(200 μL) was added to each tube, the tubes were covered and incubated at62° C. for 10 minutes. Reconstituted Enzyme Reagent (25 μL) was addedand the tubes were incubated at 42° C. for 60 minutes. Probe Reagent(100 μL) containing 2e6 RLU of Probe T was added to each tube and thetubes were incubated at 62° C. for 20 minutes followed by 5 minutes atroom temperature. Label on the non-hybridized probes was inactivated byadding 250 μL of Selection Reagent and incubating at 62° C. for 10minutes. The tubes were cooled at room temperature for 15 minutes. Thetubes were analyzed in a LEADER luminometer. Ten replicates were run foreach IVT concentration. The results are summarized in Table 7 below andthe RLU values of this table indicate that Probe T was able to detectboth of the T. vaginalis strains with similar sensitivity.

TABLE 7 Detection of T. vaginalis Strains Using Probe T ATCC No. 30001ATCC No. 30488 IVT Amt. Ave. RLU % CV Ave. RLU % CV 0 1,491 7 1,491 7 1e6 1,293,237 3 1,399,276 3 10e6 1,425,110 1 1,447,286 3 140e6 1,344,732 4 1,498,959 7

Example 5: Cross-Reactivity of T. vaginalis Detection Probe withPentatrichomonas hominis and Trichomonas tenax

In this example, Probe T (SEQ ID NO:33, AE incorporated using a linkerpositioned between bases 6 and 7) was evaluated to determine itscross-reactivity with Pentatrichomonas hominis (ATCC No. 30000) andTrichomonas tenax (ATCC No. 30207), two bacteria that are closelyrelated to T. vaginalis. Probe T was evaluated at 2e6 RLU per 100 μLusing Target Capture, TMA and HPA. The procedures and concentrationswere the same as those described in Example 3, unless otherwiseindicated. The target capture probe was SEQ ID NO:59 and theamplification oligonucleotiedes were SEQ ID Nos. 45, 53, and 61.Pentatrichomonas hominis was tested at 9.6e5 cells per test and T. tenaxwas tested at 3.8e5 cells per test. Twenty replicates were run for eachmicroorganism. Trichomonas vaginalis in vitro transcript at 1e6 copiesper test was used as the positive control and Lysis Buffer was used asthe negative control, ten replicates were run for each control. Theresults are summarized in Table 8 below and indicate that Probe T doesnot cross-react with P. hominis or T. tenax.

TABLE 8 Cross-Reactivity of Probe T Ave. RLU % CV Neg. control 1,197 8Pos. control 929,906 3 P. hominis 6,424 16 T. tenax 3,829 21

Example 6: Sensitivity of T. vaginalis Detection Probe

In this example, the sensitivity of Probe T, (SEQ ID NO:33, AEincorporated using a linker positioned between bases 6 and 7) wasevaluated using 0, 1e-7, 1e-6, 1e-5, 1e-4, 0.001, 0.01, and 1 cellequivalents of T. vaginalis lysate. Probe T was evaluated at 2.5e6 RLUper 100 μL of Probe Reagent. Probe T was evaluated using Target Capture,TMA, and HPA. The procedures and concentrations were the same as thosedescribed in Example 3, unless otherwise indicated. The capture probehad the nucleotide sequence of SEQ ID NO:59 and the amplificationoligonucleotides had the nucleotide sequences of SEQ ID Nos. 45, 53, and61. Ten replicates were run for each cell concentration. The results aresummarized in Table 9 below and indicate that the oligonucleotidecombination of SEQ ID Nos. 59, 45, 53, 61, and Probe T was able todetect the equivalent of 0.01 T. vaginalis cells.

TABLE 9 Sensitivity of the T. vaginalis Probe T T. vaginalis CellEquivalents Ave. RLU % CV 0 1,500.9 2 1e−7 1,330.9 3 1e−6 1,414.0 7 1e−52,160.9 19 1e−4 7,067.2 19 0.001 58,824.7 10 0.01 452,465.1 15 0.11,374,721 3 1 1,458,061 2

Example 7: Cross-Reactivity of T. vaginalis Detection Probe with CommonGenitourinary Bacteria

In this example, the specificity of Probe T (SEQ ID NO:33, AEincorporated using a linker positioned between bases 6 and 7), combinedwith a target capture probe (SEQ ID NO:59) and amplificationoligonucleotides (SEQ ID Nos. 45, 53, and 61), was evaluated againstseveral common urogenital bacteria. The bacteria tested were thefollowing: Chlamydia trachomatis, Neiserria gonorrhoeae, Mycoplasmagenitalium (ATCC No. 33530), Derxia gummosa (ATCC No. 15994),Enterococcus faecalis (ATCC No. 19433), Moraxella osloensis, Neiserriameningitidis (serogroups A (ATCC No. 13077), B (ATCC No. 23255), C (ATCCNo. 13109), and D (ATCC No. 13113)), Lactobacillis acidophilus (ATCC No.4356), Lactobacillis brevis (ATCC No. 14869), Lactobacillis jensonii(ATCC No. 25258), Lactobacillis lactis (ATCC No. 11454), Candidaalbicans (ATCC No. 18804), Candida glabrata (ATCC No. 48435), Candidaparapsilosis (ATCC No. 22019), Candida tropicalis (ATCC No. 750),Escherichia coli (ATCC No. 25922), Gardenerella vaginalis (ATCC No.14018), Staphylococcus aureus (ATCC No. 12598), Staphylococcuseppidermidis (ATCC No. 14990), Giardia intestinalis (ATCC No. 30888),and Ureaplasma urealyticum (ATCC No. 27618).

For each bacteria listed above, approximately 1e6 bacteria were lysed in400 μL of Lysis Buffer in a 12 mm×75 mm test tube. Target CaptureReagent (100 μL) containing 1 nmol/L of SEQ ID NO:59 was added to eachtube and the tubes were incubated at 62° C. for 30 minutes. The tubeswere then incubated for 30 minutes at room temperature. The magneticbeads in the Target Capture Reagent were pelleted using a magnetseparation unit and the supernatant was aspirated. The magnetic beadswere resuspended in 1 mL of Wash Solution, re-pelletted and the WashSolution was aspirated. The magnetic beads were resuspended in 75 μL ofreconstituted Amplification Reagent containing 53 pmol/mL of SEQ IDNO:45; 53 pmol/mL of SEQ ID Nos. 53; and 0.4.4 pmol/L of SEQ ID NO:61.Oil Reagent (200 μL) was added to each tube and the tubes were incubatedat 62° C. for 10 minutes. The tubes were then incubated at 42° C. for 5minutes. Reconstituted Enzyme Reagent (25 μL) was added to each tube andthe tubes were incubated at 42° C. for 60 minutes. Reconstituted ProbeReagent (100 μL) containing 2e6 RLU of Probe T was added to each tubeand the tubes were incubated at 62° C. for 20 minutes followed by a 5minute room temperature incubation. Selection Reagent (250 μL) was addedto each tube and the tubes were incubated at 62° C. for 10 minutes. Thetubes were then cooled at room temperature for 15 minutes. The tubeswere analyzed in a LEADER luminometer that automatically added DetectReagent I (200 μL) and Detect Reagent II (200 μL) and repeatedly readthe light emission form the tubes. The negative control did not have anybacteria or T. vaginalis in vitro transcript, the positive control had1e6 copies of T. vaginalis in vitro transcript. The controls were run induplicate, whereas the bacteria were run in triplicate. The results arelisted in Table 10 below and indicate that Probe T did not cross-reactwith the common urogential bacteria.

TABLE 10 Cross-Reactivity Against Genitourinary Bacteria ResultsBacteria Ave. RLU % CV Neg. Control 2,004 4 Pos. Control 2,247,270 1 C.trachomatis 1,859 3 N. gonorrhoeae 1,746 3 M. genitalium 1,664 5 D.gummosa 1,784 6 E. faecalis 1,706 3 M. osloensis 1,644 5 N.meningitidis, Strain A 1,715 2 N. meningitidis, Strain B 1,672 0 N.meningitidis, Strain C 1,621 20 N. meningitidis, Strain D 1,805 24 L.acidophilus 2,515 5 L. brevis 1,753 0 L. jensonii 1,618 5 L. lactis1,797 8 C. albicans 1,678 4 C. glabrata 1,588 11 C. parapsilosis 1,77844 C. tropicalis 2,421 1 E. coli 1,607 5 G. vaginallis 1,669 5 S. aureus1,732 8 S. eppidermidis 1,572 2 G. intestinalis 1,873 21 U. urealyticum1,813 5

Example 8: T. vaginalis Pseudo Target for Detuning Amplification

In this example, a pseudo target (SEQ ID NO:61) was evaluated todetermine its ability to decrease the sensitivity of the T. vaginalisamplification assay. Pseudo targets have been previously described inNunomura, U.S. Pat. No. 6,294,338, the contents of which are herebyincluded by reference herein. Briefly, a pseudo target is anoligonculeotide that is designed to compete with a target for assayresources. The pseudo target binds with a first primer to create a shortamplicon that contains a region that will bind with the second primer.The primer binding region of a pseudo target may be altered to increaseor decrease its binding affinity and thus increase or decrease theaffect of the pseudo target. The short amplicon produced from a pseudotarget does not contain a region that would allow a detection probe tobind. Pseudo targets may be created for any amplificationoligonucleotide combination. Examples of pseudo targets designed to beused with SEQ ID Nos. 45 and 53 are shown in Table 11 below.

TABLE 11  T. vaginalis Pseudo Targets SEQ ID NO. Sequence 61gctaacgagcgagattatcgccaagcaat aacaggtccgtgatg 65 ttgcttggcgataatctcgctcg66 cctgttattgcttggcgataatctcgc 67 cggacctgttattgcttggcgataatctc

A pseudo target having the nucleotide sequence of SEQ ID NO:61 wasevaluated to determine its ability to reduce the sensitivity of anoligonucleotide combination made up of a target capture probe having thenucleotide sequence of SEQ ID NO: 59, amplification oligonucleotideshaving the nucleotide sequences of SEQ ID Nos. 45 and 53, and Probe T(SEQ ID NO:33 (AE incorporated using a linker positioned between bases 6and 7). The pseudo target was evaluated using Target Capture, TMA, andHPA. The procedures were the same as those described in Example 3,however the concentrations were as follows. ATCC No. 30001 IVT wasdiluted to 5e6 copies/mL, 2e5 copies/mL, or 0 copies/mL in Lysis Buffer.The Target Capture Reagent contained 18.5 micrograms (μg) of SEQ IDNO:59. The reconstituted Amplification Reagent contained 53 nanomolar(nM) of SEQ ID NO:45; 53 nM of SEQ ID Nos. 53; and 0, 0.44, 1.3, 4.4,13, or 44 femtomoles (fmol)/mL of SEQ ID NO:61. Ten replicates were runfor each pseudo target concentration at each IVT concentration. Theresults are summarized in Table 12 below and indicate that the pseudotarget concentrations evaluated reduced the sensitivity of theoligonucleotide combination when 2e5 copies/mL of IVT were present.

TABLE 12 Pseudo Target Titration Results IVT Amt. Pseudo Target(copies/mL) Amt. (fmol/mL) Ave. RLU % CV 0 0 100,517 74 0 0.44 23,539 900 1.3 4,221 128 0 4.4 0 0 0 13 0 0 0 44 0 0 2e5 0 1,118,522 2 2e5 0.441,107,886 1 2e5 1.3 1,052,239 2 2e5 4.4 713,540 3 2e5 13 237,831 16 2e544 46,838 30 5e6 0 1,142,772 3 5e6 0.44 1,106,056 1 5e6 1.3 1,123,895 85e6 4.4 1,119,931 2 5e6 13 1,077,218 1 5e6 44 775,404 11

Example 9: Oligonucleotide Combination for Detecting Two Strains of T.vaginalis

In this example, an oligonucleotide combination made up of a targetcapture probe having the nucleotide sequence of SEQ ID NO: 59,amplification oligonucleotides having the nucleotide sequences of SEQ IDNos. 45 and 53, a pseudo target having the nucleotide sequence of SEQ IDNO:61, and a Probe T (SEQ ID NO:33, AE incorporated using a linkerpositioned between bases 6 and 7) was evaluated using two T. vaginalisstrains, ATCC Nos. 30236 and 50138. Each strain was tested at 0, 0.01,0.025, 0.05, 0.1, 1, and 5 cells per mL. The T. vaginalis cells werelysed in Lysis Buffer and 400 μL of the lysed cells were transferred toa 12 mm×75 mm tube. The T. vaginalis lysate underwent Target Capture,TMA, and HPA. The procedures and concentrations were the same as thosedescribed in Example 3, unless otherwise indicated. The threshold RLUfor a positive test was 100,000 RLU. Five replicates were run for eachlysate concentration. The results are summarized in Table 13 below andindicate that this oligonucleotide combination was able to detect T.vaginalis cells at a concentration equivalent to 0.1 T. vaginalis cellper mL.

TABLE 13 T. vaginalis Cell Line Testing Results ATCC No. 50138 ATCC No.30236 Cells/mL Ave. RLU % CV Ave. RLU % CV 0 1,511 8 1,511 8 0.01 18,83624 15,840 20 0.025 44,968 10 41,012 14 0.05 91,948 13 85,608 8 0.1164,164 11 178,063 9 1 1,029,097 23 981,508 15 5 1,599,356 2 1,533,580 3

Example 10: Oligonucleotide Combination for Detecting T. vaginalis inClinical Specimens

In this example, an oligonucleotide combination made up of a targetcapture probe having the nucleotide sequence of SEQ ID NO: 59,amplification oligonucleotides having the nucleotide sequences of SEQ IDNos. 45 and 53, a pseudo target having the nucleotide sequence of SEQ IDNO:61, and Probe T (SEQ ID NO:33, AE incorporated using a linkerpositioned between bases 6 and 7) was evaluated using two types ofclinical specimens spiked with T. vaginalis cells. The first type ofclinical specimen used was urine samples that were collected from 32adult females. The urine samples were pooled and diluted 1:1 with UrineLysis Buffer (ULB). The second type of clinical specimen used wascervical samples collected in ThinPrep® media (Hologic, Inc.;Marlborough, Massachusettes). The ThinPrep samples were pooled anddiluted 1:2.9 with Lysis Buffer (LB). Trichomonas vaginalis cells (ATCCNo. 30236) were lysed in Lysis Buffer and spiked into the urine-ULB orThinPrep-LB to a final concentration of 1, 0.3, 0.1, 0.03, 0.01, 0.003,0.001, and 0 cells per milliliter of urine-ULB or ThinPrep-LB. Thespiked clinical specimens underwent Target Capture, TMA and HPA. Theprocedures and concentrations were the same as those described inExample 3, unless otherwise indicated. Thirty replicates were run foreach clinical specimen type at each spike concentration and fivereplicates were run for each clinical specimen type without spiking. Theresults are summarized in Table 14 below and indicate that theoligonucleotide combination was able to detect the equivalent of 0.03 T.vaginalis cells/mL in either a urine specimen or a ThinPrep specimen.

TABLE 14 Clinical Specimens Results Urine ThinPrep Cells/mL Ave. RLU %CV Ave. RLU % CV 0 0 N/A 0 N/A 0.001 3,414 153 1,292 236 0.003 29,715 3112,546 61 0.01 120,574 15 75,762 18 0.03 331,695 10 198,820 14 0.1754,571 5 527,972 6 0.3 1,048,161 5 874,054 6 1 1,096,693 5 1,016,130 5

Example 11: Stability of T. vaginalis in Cervical Cells Collected inLiquid Based Cytology Media

In this example, an oligonucleotide combination made up of a targetcapture probe having the nucleotide sequence of SEQ ID NO: 59,amplification oligonucleotides having the nucleotide sequences of SEQ IDNos. 45 and 53, a pseudo target having the nucleotide sequence of SEQ IDNO:61, and Probe T (SEQ ID NO:33, AE incorporated using a linkerpositioned between bases 6 and 7) was evaluated using cervical cellscollected in SurePath® (Becton Dickinson; Franklin Lakes, N.J.) andThinPrep liquid based cytology media. Cervical samples that werenegative for T. vaginalis were pooled together and spiked with T.vaginalis cells to a final concentration of 1,000 cells per 400 μL ofeither SurePath or ThinPrep media. The spiked pools were stored at 30°C. Aliquots of 1 mL were removed from the spiked cervical sample poolsafter 0, 1, and 2 days. The aliquots were added to 2.9 mL of LysisBuffer. The lysed aliquots were diluted down to 0.1, 1, 10, and 100cells per 400 μL. Due to the limited nature of the samples, non-spikedsamples were not tested.

The SurePath media material safety data sheet lists formaldehyde, whichis known to cross-link and degrade nucleic acid. To help reverse theaffects of the formaldehyde, some of the aliquots taken from theSurePath sample pool were further treated with FAST Express Reagent(Gen-Probe Cat. No. 102930) before diluting down to 0.1, 1, 10, and 100cells per 400 μL. For the SurePath sample pool, two aliquots wereremoved for the day 1 and 2 time points. One of the aliquots was treatedwith FAST Express Reagent, which consisted of reconstituting thelyophilized reagent with 1 mL of water, adding 100 μL of thereconstituted reagent to the aliquots, and incubating the aliquots at65° C. for 2 hours.

The diluted samples underwent Target Capture, TMA and HPA. Theprocedures and concentrations were the same as those described inExample 3, unless otherwise indicated. Ten replicates were run for eachdilution level at each time point. The results are summarized in Table16 below and indicate that the oligonucleotide combination was able todetect the samples collected in ThinPrep media at all dilution levelsover the three time points, but was only able detect the specimenscollected in SurePath at the 10 or 100 cell dilution level after oneday.

TABLE 16 Results for the Liquid Cytology Samples Day 0 Day 1 Day 2Sample Description Ave. RLU % CV Ave. RLU % CV Ave. RLU % CV SurePath100 cells 1,227,100 2 1,193,900 3.66 1,171,000 3.88 SurePath 10 cells1,226,300 3 604,000 16.95 298,100 6.40 SurePath 1 cell 1,178,200 552,900 24.17 34,000 20.84 Surepath 0.1 cell 468,000 8 6,900 62.78 4,90032.55 SurePath 100 cells + NT NT 1,016,400 2.26 992,100 3.85 FastExpress SurePath 10 cells + NT NT 248,800 5.91 227,900 9.00 Fast ExpressSurePath 1 cell + NT NT 27,000 15.91 27,100 13.30 Fast Express Surepath0.1 cell + NT NT 6,000 38.49 5,400 30.49 Fast Express ThinPrep 100 cells1,219,400 3 1,254,000 4.99 1,254,800 4.17 ThinPrep 10 cells 1,250,800 21,239,200 4.29 1,206,200 3.11 ThinPrep 1 cell 1,209,600 2 958,000 2.90859,300 4.62 ThinPrep 0.1 cell 754,300 2 209,900 8.62 166,200 10.14 “NT”means not tested

Example 12: New Target Capture Probes for Detecting T. vaginalis inSamples Collected in SurePath Media

In this example, several new target capture probes (SEQ ID NO:89, SEQ IDNO:94, SEQ ID NO:99, SEQ ID NO:104, SEQ ID NO:105, and SEQ ID NO:106)were evaluated in two different experiments. The performance of SEQ IDNO:59 in samples collected in SurePath media prompted the development ofnew target capture probes that would perform better with samplescollected in SurePath media. The new target capture probes were comparedto SEQ ID NO:59.

In the first experiment, SEQ ID NO:89 (first 22 bases were 2′-O-MethylRNA), SEQ ID NO:94, SEQ ID NO:105 (first 24 bases were 2′-O-Methyl RNA),and SEQ ID NO:106 (first 19 bases were 2′-O-Methyl RNA) were evaluatedagainst SEQ ID NO:59. Trichomonas vaginalis cells were tested at 0.2cells per mL of Lysis Buffer. The new target capture probes were testedat 0.5 and 1 pmol per 100 μL of Target Capture Reagent. SEQ ID NO:89,SEQ ID NO:105, and SEQ ID NO:106 were evaluated as 2′-O-Methyl RNAoligonucleotides, whereas SEQ ID NO:94 and SEQ ID NO:59 were evaluatedas DNA oligonucleotides. The target capture probes were evaluated usingamplification oligonucleotides having the nucleotide sequences of SEQ IDNos. 45 and 53, a pseudo target having the nucleotide sequence of SEQ IDNO:61, and Probe T (SEQ ID NO:33, AE incorporated using a linkerpositioned between bases 6 and 7). The samples underwent Target Capture,TMA and HPA. The procedures and concentrations were the same as thosedescribed in Example 3, unless otherwise indicated. Twenty replicateswere run for each target capture probe. The results are summarized inTable 17 below and indicate that SEQ ID NO:94, SEQ ID NO:105, and SEQ IDNO:106 perform similar to or better than SEQ ID NO:59.

TABLE 17 Results for the New Target Capture Probes, First ExperimentTarget Capture Probe 0.2 cells/ml and Amount Ave. RLU % CV SEQ ID NO: 59296,572 25 SEQ ID NO: 89 at 1 86,273 12 SEQ ID NO: 94 at 1 304,351 9 SEQID NO: 105 at 1 893,675 8 SEQ ID NO: 106 at 1 810,486 8 SEQ ID NO: 89 at0.5 141,617 15 SEQ ID NO: 94 at 0.5 224,187 10 SEQ ID NO: 105 at 0.5732,092 15 SEQ ID NO: 106 at 0.5 645,401 6

In the second experiment, SEQ ID NO:99 and SEQ ID NO:104 were evaluatedagainst SEQ ID NO:59. The new target capture probes were evaluated at 1pmol per 100 μL of Target Capture Reagent. Trichomonas vaginalis invitro transcript was tested at 0, 2e5 and 1e6 copies per mL of LysisBuffer and T. vaginalis cells were tested at 0 and 13 cells per mL ofLysis Buffer. The target capture probes were evaluated usingamplification oligonucleotides having the nucleotide sequences of SEQ IDNos. 45 and 53, a pseudo target having the nucleotide sequence of SEQ IDNO:61, and a Probe T (SEQ ID NO:33, AE incorporated using a linkerpositioned between bases 6 and 7). The samples underwent Target Capture,TMA and HPA. The procedures and concentrations were the same as thosedescribed in Example 3, unless otherwise indicated. Eight replicateswere run samples containing 0 or 2e5 copies of in vitro transcript and12 replicates were run for samples containing 1e6 copies of in vitrotranscript. Two replicates were run for the samples containing cells.The results are summarized in Table 18 below and indicate that SEQ IDNO:99 and SEQ ID NO:104 perform poorer than SEQ ID NO:59 at low levelsof detection.

TABLE 18 Results for the New Target Capture Probes, Second ExperimentTarget Capture Probe, Sample Ave RLU % CV SEQ ID NO: 59, 0 cells 2,50085 SEQ ID NO: 59, 13 cells 1,244,000 3 SEQ ID NO: 59, 0 IVT 2,625 40 SEQID NO: 59, 2e5 IVT 1,008,125 6 SEQ ID NO: 59, 1e6 IVT 393,333 9 SEQ IDNO: 99, 0 cells 2,000 0 SEQ ID NO: 99, 13 cells 876,500 13 SEQ ID NO:99, 0 IVT 3,250 32 SEQ ID NO: 99, 2e5 IVT 11,625 32 SEQ ID NO: 99, 1e6IVT 4,833 36 SEQ ID NO: 104, 0 cells 3,500 61 SEQ ID NO: 104, 13 cells130,108 13 SEQ ID NO: 104, 0 IVT 3,125 63 SEQ ID NO: 104, 2e5 IVT 23,25029 SEQ ID NO: 104, 1e6 IVT 6,917 37

Example 13: Dual-Target Capture Probes for Detecting T. vaginalis inCervical Cells Collected in SurePath Media

In this example, an oligonucleotide combination made up of two targetcapture probes having nucleotide sequences of SEQ ID NO: 59 (at 0.1pmol/100 μL) and SEQ ID NO:94 (at 1.5 pmol/100 μL), amplificationoligonucleotides having the nucleotide sequences of SEQ ID Nos. 45 and53, a pseudo target having the nucleotide sequence of SEQ ID NO:61, andProbe T (SEQ ID NO:33, AE incorporated using a linker positioned betweenbases 6 and 7) was evaluated using cervical cells collected in SurePathliquid based cytology media. Cervical samples that were negative for T.vaginalis were spiked with T. vaginalis cells to a final concentrationof 1,000 cells per 400 μL of SurePath media. Aliquots of 500 μL wereremoved from the spiked cervical samples at 0 and 3 days and added to2.9 mL of Lysis Buffer. The lysed samples were treated with 100 μL ofFAST Express Reagent that was reconstituted in 1 mL of water. Thesamples were incubated at 65° C. for 2 hours. Following the incubation,the samples were diluted to 1, 10, and 100 cells per 400 μL. The dilutedsamples underwent Target Capture, TMA and HPA. The procedures andconcentrations were the same as those described in Example 3, unlessotherwise indicated. The day 3 samples were also tested using theoligonucleotide combination described in Example 11 (SEQ ID Nos. 45, 53,59, 61, and Probe T). Four replicates were run for each clinicalspecimen at the three dilution levels for the day 0. Three replicateswere run for all others. The results are summarized in Table 19 belowand indicate that the dual-target capture oligonucleotide combinationperformed better than single target capture oligonucleotide combinationat detecting 1 cell per 400 μL.

TABLE 19 Results for Single and Dual Target Capture Probes Single TargetCapture Probe Dual Target Capture Probes Sample ID & Day 3 Day 0 Day 3Description Ave RLU % CV Ave RLU % CV Ave RLU % CV 1 at 100 cells1,174,667 1 1,385,000 3 1,435,333 0 1 at 10 cells 219,000 7 1,270,750 21,079,333 4 1 at 1 cell 26,667 16 1,250,000 2 217,667 9 1 at 0 cells NTNT 1,333 43 NT NT 2 at 100 cells 1,313,333 6 1,372,667 1 1,443,333 1 2at 10 cells 1,262,000 2 1,277,000 2 1,421,667 4 2 at 1 cell 380,000 61,267,500 0 1,409,333 2 2 at 0 cells NT NT 1,333 43 NT NT 3 at 100 cells1,399,000 2 1,407,500 3 1,428,333 2 3 at 10 cells 889,000 5 1,269,000 21,445,000 2 3 at 1 cell 1,667 35 1,265,250 2 1,667 35 3 at 0 cells NT NT1,667 69 NT NT 6 at 100 cells 1,387,667 1 1,377,000 4 1,468,667 1 6 at10 cells 1,019,667 3 1,288,000 2 1,450,000 3 6 at 1 cell 22,333 91,252,250 2 1,409,000 2 6 at 0 cells NT NT 2,667 43 NT NT 8 at 100 cells995,000 1 1,418,667 2 1,409,667 2 8 at 10 cells 183,000 15 1,296,000 11,246,333 1 8 at 1 cell 22,333 9 1,274,500 1 330,667 8 8 at 0 cells NTNT 1,000 0 NT NT 9 at 100 cells 1,152,667 0 1,394,667 2 1,441,000 2 9 at10 cells 221,333 7 1,275,750 3 1,339,667 2 9 at 1 cell 29,000 271,256,750 3 486,000 6 9 at 0 cells NT NT 1,667 35 NT NT 10 at 100 cells1,334,667 2 1,357,333 1 1,433,667 2 10 at 10 cells 486,000 11 1,272,7501 1,161,667 1 10 at 1 cell 2,000 0 1,262,000 2 383,000 2 10 at 0 cellsNT NT 1,333 43 NT NT 13 at 100 cells 1,124,333 8 1,369,667 2 1,365,000 413 at 10 cells 219,000 10 1,266,000 1 972,000 2 13 at 1 cell 20,667 71,252,500 3 164,000 9 13 at 0 cells NT NT 6,333 40 NT NT “NT” means nottested

While the disclosure has been described and shown in considerable detailwith reference to certain embodiments, those skilled in the art willreadily appreciate other embodiments of the disclosure. Accordingly, thedisclosure is deemed to include all modifications and variationsencompassed within the spirit and scope of the following appendedclaims.

The invention claimed is:
 1. A set of capture probes comprising: a firstcapture probe consisting of: (i) a target-complementary sequence, thebase sequence of which is selected from the group consisting of the basesequences of SEQ ID NO: 90 and SEQ ID NO: 91; and (ii) anon-complementary sequence comprising a homopolymer tail, wherein thenon-complementary sequence does not stably bind to a target nucleic acidderived from Trichomonas vaginalis when the target-complementarysequence is stably hybridized to the target nucleic acid; and a secondcapture probe consisting of: (i) a target-complementary sequence, thebase sequence of which is selected from the group consisting of SEQ IDNO: 55 and SEQ ID NO: 56; and (ii) a non-complementary sequencecomprising a homopolymer tail, wherein the non-complementary sequencedoes not stably bind to a target nucleic acid derived from Trichomonasvaginalis when the target-complementary sequence is stably hybridized tothe target nucleic acid.
 2. The set of capture probes of claim 1,wherein the non-complementary sequence of each of the first and secondcapture probes comprises a spacer region for joining thetarget-complementary sequence to the homopolymer tail.
 3. The set ofcapture probes of claim 1, wherein the homopolymer tail of each of thefirst and second capture probes is a poly dA sequence.
 4. The set ofcapture probes of claim 2, wherein the homopolymer tail of each of thefirst and second capture probes is a poly dA sequence, and wherein thespacer region of each of the first and second capture probes is a polydT sequence.
 5. The set of capture probes of claim 1, wherein thetarget-complementary sequence of each of the first and second captureprobes comprises at least one ribonucleoside having 2′-O-methylsubstitution to the ribofuranosyl moiety.
 6. The set of capture probesof claim 2, wherein the non-complementary sequence of each of the firstand second capture probes consists of the homopolymer tail and thespacer region.
 7. The set of capture probes of claim 3, wherein thenon-complementary sequence of each of the first and second captureprobes consists of the homopolymer tail.
 8. The set of capture probes ofclaim 4, wherein the non-complementary sequence of each of the first andsecond probes consists of the homopolymer tail and the spacer region. 9.The set of capture probes of claim 1, wherein the non-complementarysequence of each of the first and second probes consists of the sequenceof SEQ ID NO:60.